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Biologia molecolare applicata alle biotecnologie, Biologia molecolare, inglese

Programma
Non-coding regulatory rnas
- Small non-coding RNAs (snRNA, snoRNA, siRNA, microRNA, piRNA): definition, classification, structure, mechanism of action and physio-pathological examples.
- Long non-coding RNAs: definition, classification, structure, mechanism of action and physio-pathological examples.

Dynamics of the repetitive elements of dna in cell identity, differentiation,... Vedi di più

Esame di Molecular biology applied to biotechnology docente Prof. M. Pagani

Anteprima

ESTRATTO DOCUMENTO

! After! just! few! months’! people! started! to! enhance! this! technology,! and! the! first! really! big!

1. Acquisition'→'When!one!phage!is!infecting!one!bacterial!cell,!there!are!some!elements,! advance!they!obtained!was!the!discovery!that!is!possible!to'fuse'the'crRNA'with'the'tracrRNA,!

some!nucleases!that!are!able!to!recognize!the!phage!DNA,! to! cleave!its!sequence!and! obtained!the!so!called!“Guide=RNA”.!

then,!to!integrate!it!(shown!in!the!figure!with!green)!into!this!array.!!!

2. Expression'→'Then!the!CRISPR!array!(and!the!green!sequence)!is!transcribed,!and!its!

transcription!produces!the!so!called!“pre=crRNA”!that!is!built!by!repetition!of!hairpin!

loops,! that! are! the! common! repeated! sequences,! with! the! protospacers,! that! are! the!

sequences'derived'from'the'phage.!!

This!RNA!is!then!processed,!obtaining!several!crRNAs!that!contain!a!short!sequence!of!

RNA!that!is!complementary!to!the!phage’s!DNA.!!

Basically! these! crRNAs! are! able! to! target'other'proteins,! and! one! of! these! proteins! is!

belonging!to!Cas!proteins!that!are!endonucleases.!!

3. Interference'→!those!Cas!endonucleases,!thanks!to!the!RNA!sequence!derived!from!the! !

phage,!are!able!to!recognize!the!complementary!sequence!into!the!phage’s!genome!and! So! it! is! possible! to! fuse! these! two! molecules,! obtaining! only! one! molecule! of! RNA,! and! the!

cleave!it.!! interesting!thing!is!that!in!this!molecule!of!RNA!that!is!about!hundreds!of!nucleotides,!the!only!

! variable'part'are'the'20'nt!of!the'original'crRNA.!

So!this!crSRNA!is!able!to!induce!the!targeting!and!the!cleavage!of!phage’s!DNA.!! We!can!have!a!plate!mate!containing!the!backbone!of!one!guideSRNA,!and!we!can!simply!clone!

This!system!reminds!in!some!way!the!small!RNA!system.!! 20! nt,! that! is! very! simple! to! obtain! producing! two! oligonucleotides,! and! ligate! this! two!

! oligonucleotides!in!a!vector.!!!

There! are! several! types! of! CRISPR! system! in! different! bacteria.! The! most! characterized! The!production!of!a!guide!RNA!is!very!easy!to!obtain.!

"

CRISPR! system! is! typeII.! In! this! system! we! have! also! another! transcript! in! the! opposite! !

orientation! that! contains! one! common! element.! In! contrast! to! other! CRISPR! system,! Cas9! is! The!second!optimization!of!this!system!was!the!creation!of!a!humanized!Cas9!protein!that!can!

the!only!component!in!inference!complex!in!TypeII!CRISPR!system.!! be!expressed!also!in!mammalian!cells.!!

! '

* The! sequence! of! the! guideSRNA! is! fundamental! for! function! of! the! Cas9;! therefore,! the! Cas9!

* without!the!guideSRNA!is!not!properly!folded.!!

Cas9:*RNA*directed*endonuclease* The!variable!part!of!the!guide!RNA!is!the!only!part!fundamental!for!the!cleavage.!

!

The!PAM!sequence!!

Cas! is! an! endonuclease,! and! it! is! able! to! cleave! a! particular! sequence! to! destroy! the! phage!

Common( itself.!!

part((( The! region! that! is! targeted! by! the! guideSRNA! has! to! be! followed! by! the! soScalled! “PAM!

sequence”!that!is!different!for!the!different!Cas!enzymes!in!the!different!bacteria.!!

Variable( In!Cas!9,!the!PAM!sequence!is!obtained!by!the!sequence!NGG:!any!nucleotide!followed!by!two!

part(( guanines.!

* So!basically!the!whole!sequence!that!is!fundamental!for!the!recognition!is!the!20!nt!plus!the!

The!crRNA!contains!two!portions:! PAM!region.!!

Variable'portion'→!confers!the!specificity!and!it!is!complementary!to!the!target!DNA!

• !

Common'portion!→!that!is!complementary!to!the!tracrRNA.!!

• Cas' is' able' to' cleave' this' sequence' into' the' phage’s' genome' and' induce' the' insertion' of' this'

This!TransSCRISPRSRNA!contains!also!another!part!that!is!fundamental!for!the!folding!of!this! sequence'into'the'genome'of'the'bacteria'that'do'not'contain'the'PAM'sequence.'

Cas9!protein.! The!PAM!region!is!not!cleaved!and!inserted!into!the!bacterial!genome!and!this!is!why!the!Cas9!

So,!these!two!RNA!sequences!are!enough!to!induce!the!specificity!of!targeting!of!the!Cas!9.! doesn’t!cleave!the!CRISPR!array!into!the!bacterial!genome.!

In!addition,!the!Cas9!protein!contains!two!nucleases'domain!able!to!cleave!the!DNA!resulting! Cas!enzymes!that!recognize!this!sequence!in!the!phage!remove!only!this!20nt!and!insert!them!

in!a!blunt!cleavage!of!the!molecule!of!DNA.! in!the!array.!!

! In!this!way,!we!do!not!have!the!cleavage!of!the!bacterial!chromosome.!!

Cas9*as*RNA=dependent*programmable*DNA*Endonuclease** !

In!2012!was!published!the!first!paper!about!Cas9!as!an!RNA!dependent!programmable!DNA! Therefore! PAM! sequence! is! fundamental! for! the! recognition! and! for! avoiding! that! the! Cas9!

endonuclease.! cleaves!the!bacterial!genome.!

This! was! the! first! demonstration! that! with! the! CRISPR! system! was! possible! to' create' an'

artificial'restriction'enzyme,'which'specificity'derives'by'one'RNA'molecule.!!

'

! 47! ! 48!

So!at!the!end!of!the!day!the!PAM!sequence!is!a!fundamental!part!of!the!consensus!sequences! Considering! the! efficiency! of! mutation! for! different! alleles,! with! this! system! it! is! possible! to!

recognized! by! the! CRISPRSCas9! system,! but! is! not! recognized! by! the! interaction! of! the! RNA! obtain!a!60?70%'of'success'in'double'knock?outs!in!the!first!round!of!production.!!

versus!the!DNA.!! !

! The! difference! is! 3! weeks! compared! to! 3! years! so! this! system! completely! changed! the!

! possibility!to!obtain!new!model!animals.!

Crisp\cas9!as!genome!editing!tools! !

In!2014!a!couple!of!papers!described!the!creation!of!the!humanized!Cas9!and!the!creation!of! Knock*in*generations*

the!guide!RNA,!and!they!demonstrated!that!this!programmable!enzyme!is!able!to!work!also!in! It!is!also!possible!to!use!the!CRISPRSCas9!approach!to!knockSin!and!to!perform!specific!and!

human!cells.!! targeted!mutations.!!

Starting!to!2012!until!now,!a!lot!of!papers!have!been!published.!! This!is!based!on!the!injection!of!the!Cas9,!the!guideSRNA!and!single!strand!oligoNTs!or!vectors!

! and!the!action!of!the!HomologySdependent!repair.!

Knock!out!mouse!modified!multiple!locus!with!single!step! !

The! group! of! Rudolf! Jaenisch! described! the! exploitation! of! the! CRISPRSCas9! system! for! the! !

editing!of!one!genome!by*knock!out!of!multiple!locus!in!a!single!step.! Example!of!modification!

! '

! In!one!month!of!work,!we!can!obtain!one!mouse!in!

Canonical*KO*system*vs*CRISPR=Cas9*approach** which!we!have!inserted!a!cassette!and!performed!

It! is! fundamental! to! have! an! idea! of! the! differences! in! time! and! cost! between! the! canonical! this!targeted!mutation.!!

system!and!the!CRISPRSCas9!based!approach.!! !

! *

Traditional'way'' *

1. When! we! want! to! perform! a! KO! system! with! the! canonical! way,! we! have! to! start! *

designing!and!creating!the!recombination!cassette,!and!this!can!require!different!days! Advantage*of*CRISPR\Cas9*over*TALEN*or*ZFN*

or!weeks.! The! real! advantage! of! CRISPSCas9! over! TALEN! or! Zinc! finger! is! due! to! the! simplicity! of! the!

2. Then!we!have!to!inject!this!plasmid!into!the!embryonic!stem!cells.! exploitation! of! the! CRISPRSCas9:! indeed! for! TALEN! or! Zinc! finger! we! have! to! create! new!

3. In! the! best! experimental! conditions,! minimum! 3! months! are! necessary! to! obtain! ES! recombinant!proteins!with!very!complex!structure.!!

cells!with!recombination!! Even! considering! TALEN,! that! is! simpler! than! the! Zinc! finger! approach,! it! is! necessary! to!

4. Then!we!have!to!inject!these!ES!into!the!blastocyst! spend! several! months! in! cloning.! This! is! because! the! structure! of! the! system! (the! array! is!

5. Add!this!blastocyst!to!the!mouse.!! obtained! by! domains! that! are! basically! identical! repetitiona! except! for! the! two! internal! aa),!

6. Wait!for!the!birth!of!the!new!mouse!that!is!chimeric.!! and!performing!a!cloning!of!this!array!of!15\16!portion!of!DNA!that!are!basically!identical!is!

7. After! this,! we! have! to! perform! different! step! of! breeding! in! order! to! obtain! very!complex!and!difficult!to!obtain.!!!

heterozygote!animals.! Also! only! the! elaboration! process! leading! to! the! production! of! simply! one! Talen! requires!

8. By!the!breeding!of!these!heterozygotes,!we!can!obtain!homozygotes.! many!days.!

We!can!spend!one!year!to!perform!one!KO!and!to!obtain!the!first!founder!mouse,!but!then!we! !

need!to!expand!the!colony.!! Validation'of'constructed'TALEN! →! the! other! problem! is! that! we! have! to! test! the! function! of!

So,!with!some!luck!and!in!the!best!conditions,!almost!1!years!is!necessary!to!obtain!the!first! both!TALEN!and!Zinc!finger.!We!cannot!be!sure!at!100%!that!the!DNA!binding!domains!are!

colony.! able!to!recognize!properly!the!DNA.!!

Then,!if!we!have!to!analyze!different!genes,!we!have!to!perform!different!crossings!between! !

the!animals,!in!order!to!obtain!the!multiple!KO!and!this!means!years!of!works.!! In'CRISPR?Cas9'system!→!In!CRISPRSCas9!system,!we!have!simply!to!synthetize!this!20nt.!!

After!all,!the!time!needed!for!the!entire!process!is!at!least!2=3*years*of*work.!!! The! only! limitation! is! that! these' 20nt' have' to' be' close' to' PAM' sequence,! and! so! the! only!

! limitation!to!the!exploitation!for!the!system!into!one!genome!is!the!presence!of!enough!PAM!

CRISPR?Cas9'system' sequences,!but!the!PAM!sequences!are!simply!a!couple!of!G!and!they!are!usually!present!with!

1. In!1!day!it!is!possible!to!design!the!oligonucleotides!of!interest!! high!frequency!in!the!genome.!!

2. In!1!week!we!can!clone!them!in!vectors!! So!the!space!for!this!modification!is!really!high.!!

3. Then!we!can!inject!these!oligos,!the!Cas9!plus!the!guideSRNA!into!a!zygote!directly.! !

Because! we! are! injecting! into! a! zygote,! the! first'animal'is! obviously! a! not! chimerical! CRISPR?Cas9'has'higher'efficiency'than'TALEN'

animal,!but!the'final'founder.! Considering!a!comparison!between!Cas!9!and!TALEN,!we!have!an!improvement!of!20!times!of!

Therefore,! in! less* than* 3* weeks! we! can! have! the! first! generation! of! founder! animals.! In! efficacy!of!homology!recombination,!that!is!the!most!difficult!application.!!

addition,! the! most! important! thing! is! that! we'can'perform'simultaneously'the'KO'of'different' Basically! the! same! sequence,! targeted! with! TALEN! or! Cas9! system,! gives! respectively! less!

genes.'' than!0.5!%!of!cells!that!are!modified!respect!80%.!!

! 49! ! 50!

Applications*of*CRISPR=Cas9! This!group!was!able!to!produce!organoids!from!the!intestine!of!patients!with!cystic!fibrosis.!!

! KnockSout\in!animal!generation! In! particular,! these! patients! have! a! typical! mutation,! the! ∆F508' in' CFTR' gene:! this! deletion!

induces!the!incorrect!folding!of!the!protein!that!doesn’t!move!to!the!membrane.!!

! Gene!knock!out!in!cultured!cell!line!!

! Gene!activation!\!repression!by!Cas9! This!protein!is!fundamental!for!the!correct!excretion!of!salts!and!mucus!into!external!part!of!

! Therapeutic!application! epithelial! tissue! and! indeed,! cystic! fibrosis! patients! have! awful! problems! in! the! lungs! and!

intestine.! The! most! critical! problems! are! in! the! lungs,! because! this! altered! balance! between!

! Others!

! the!salts!reduces!the!presence!of!innate!elements!fundamental!for!destroying!bacteria.!!

Knock'out\in'in'“other'animals”' !

This! group! demonstrated! that,! working! with! these! intestinal! stem! cells,! they! were! able! to!

The!first!clear!difference!respect!to!the!past!is!that!with!CRISPRSCas9!it!is!possible!to!perform!

knockSout!and!knockSin!also!in!different!animals.!! obtain!defective!organoids,!that!are!not!swelling!in!the!correct!way.!!

However,!they!were!able!to!repair!the!mutation,!obtaining!completely!functional!organoids.'

Until!2013,!the!only!mammalian!in!which!we!could!perform!knock!out!and!knock!in!was!the!

mouse.! Now! we! can! basically! perform! them! in! all! the! animals! (Zebrafish,! Drosophila,! Rat,! !

Rabbit...)!and!also!plants!and!bacteria…!! Now!there!are!groups,!that!are!trying!to!use!this!approach!to!treat!models!for!the!moments.!

This! can! be! a! potential! situation! in! which! we! can! purify! the! stem! cell! from! an! individual,!

!

Application'of'CRISPR?Cas9'system'in'monkeys' correct!the!mutation,!and!reSinject!the!stem!cells,!obtaining!a!potential!repair!also!for!somatic!

tissues,!fundamental!for!a!possible!future!cure!for!patients.!

Gene!targeting!of!a!monkey.!Monkeys!are!used!as!animal!models!for!studying!psychiatric!and! !

neurological!studies.!!

This!was!the!first!example!of!genome!engineering!in!primates.! The! intersection! between! the! creation! of! stem! cells! organoids! and! the! CRISPRSCas9! system!

has!a!lot!of!potential.!

!!

! !

Organoids*and*CRISPR=Cas9* Genome'scale'CRISPR?Cas9'knock'out'screening'in'human'

cells''

Functional' repairs' of' CTFR' by' CRISPR\Cas9' in' intestinal' stem' cell' organoids' of' cystic' fibrosis'

patients.'! People! also! started! to! use! the! CRISPRSCas9! to! perform!

genome!scale!screening,!in!which!we!have!the!activation!

or! the! inactivation! of! a! gene! of! interest,! so! inhibitory!

screening!or!activatory!screening.!!

The! reason! of! this,! is! connected! to! the! facility! of! the!

experiment,! producing! an! array! of! amazing! number! of!

GuideSRNA! and! clone! them! in! a! library! of! lentiviral!

vector!for!example.!!

Then!it!is!possible!to!use!this!library!of!lentiviral!vectors!

to!start!a!screening!of!gene!of!interest!or!what!ever!we!

want!to!study.!!

! !

!

The!big!discovery!was!the!identification!of!a!marker,!Lgr5!that!is!sulfate!marker!that!defines!a!

compartment!of!intestinal!stem!cells.!The!definition!of!this!marker!allowed!the!purification!of! !

intestinal!stem!cells.!! !

After!few!years,!they!were!also!able!to!do!something!else:!starting!from!this!compartment!of! !

stem!cells,!they!were!able!to!obtain!a!3D!culture!using!a!matrix!of!matrigel.! Now!we!have!different!libraries!of!GuideSRNAs!for!whole!human!genes,!that!are!available!for!

This! 3D! culture! was! organized! by! real! crypts,! so! we! have! the! differentiation! of! these! stem! every!experiment!we!want!to!perform.!

cells!that!were!able!to!mimic!completely!the!original!ones.! !

So,! considering! the! structure! of! crypt:! we! can! found! some! Lgr5+! cells! (the! intestinal! stem! Dcas9?mediated'endogenous'gene'activations''

cells),! then! other! cells! under! the! crypt! and! around! the! crypt,! that! are! other! specific! type! of! It! is! possible! to! perform! not! only! the! inhibitory! screening! but! also! screening! in! which! we!

differentiated!cells,!like!the!Paneth!cells.!! induce!the!activation!of!the!production!of!one!gene.!This!screening!is!basically!based!on!the!

These!cells!are!completely!differentiated!and!they!are!functional,!organized!in!3D!and!able!to! exploitation! of! a! mutant! version! of! Cas9,! a! defective' Cas9' (dCas9),! that! contains! two! single!

secrete!hormones!and!the!canonical!mucus!present!in!the!epithelium!and!so!on...! mutations.!These!mutations!induce!the!lack!of!functionality!in!cleavage!of!DNA:!therefore,!this!

This!is!a!very!important!discovery,!and!now!it!is!possible!to!create!different!organoids,!also! dCas9!is!not!able!to!cleave!the!DNA!but!it!has!in!any!case!a!specific!DNA!binding!domain.!!

for!lungs!and!colon.! This!is!the!trick!to!create!an!activation!screening,!so!we!can!decide!to!activate!whatever!we!

! want!in!the!whole!genome!and!perform!high!throughput!screening.!!

! 51! ! 52!

GG17CH14-Qi ARI 9 May 2016 21:41

Overview of CRISPR/Cas9 applications

! Strategies!for!improving!the!DNA!specificity!of!CRISPR–based!agents!

Overview*of*CRISPR\Cas9*applications* The! huge! trick! to! obtain! a! very! specific! gene! editing! is! the! exploitation! of! the! so! called!

“nickase”:!

Fluorescent

protein The!Nickase!is!a!defective!dCas9,!in!which!only!one!domain!is!mutated,!so!it!is!able!to!cleave!

dCas9

Cas9 only!one!strand!of!DNA.!! Double-Nicking System

Gene editing Genome imaging Exploiting! two! Cas9! that! are! driven! by! two! GuideSRNA! that! are! on! the! two! strands! of! DNA!

CRISPR/Cas9 applications very!close!to!each!other,!it!is!possible!to!obtain!a!double!cleavage!that!is!basically!driven!by!

(Versatile RNA-guided

DNA-targeting platform)

Transcription Epigenetic two! different! sequences.! So! now,! instead'of'20nt,'here'the'specificity'is'given'by'40nt:! this! is!

factor modifier really!a!unique!site!in!the!genome,!enhancing'the'specificity'1000'times.!!

dCas9 dCas9 - Using Cas9 Nickase (Can cleave only single strand of DNA

The! problem! is! that! using! this! approach! the! homology! recombination! is! a! little! bit! reduced,!

*

Gene regulation Epigenetic modification and!the!efficiency!of!this!system!is!a!little!bit!reduced!but!safety!is!more!important.!

! Canonical'gene'editing''

Figure 1

Overview of CRISPR/Cas9 applications. This system has been adapted and developed for gene editing,

! Several'application'of'gene'regulation''

transcription regulation, chromosome imaging, and epigenetic modification. Gene editing is based on the

nuclease activity of Cas9, whereas the three other applications use the catalytic, nuclease-deactivated form of

! Genome'imaging!→!if!we!create!a!chimera!of!dCas9!with!a!fluorescent!protein,!we!can!

Cas9 (dCas9). Fusing dCas9 to various effector domains enables the sequence-specific recruitment of

transcription regulators for gene regulation, fluorescent proteins for genome imaging, and epigenetic

use!this!system!to!visualize!a!portion!of!our!DNA.!We!can!follow!one!locus!of!DNA!in!

modifiers for epigenetic modification.

live!imaging,!because!we!don’t!need!to!broke!the!cells,!insert!probe!and!so!on.!We!can! - Reduces o

use! simply! the! Cas9! that! is! defective,! so! it! is! absolutely! safe,! but! it! is! able! to! bind! a!

discoveries paved the way for the use of CRISPR/Cas9 for genome engineering, including gene

editing and gene expression regulation, epigenetic modification, and genome imaging, as detailed

portion!of!DNA,!and!we!can!follow!in!time!the!movement!of!this!portion!of!DNA.!!!

1).

below (Figure ! by 50-1,00

!

! Epigenetic'modification!→!using!a!dCas9!we!can!create!new!chimeric!proteins!able!to!

2. TARGETED GENOME EDITING WITH CRISPR/CAS9

modify!the!DNA!and!histones!also!in!a!very!precise!and!programmable!way.!! Another!important!fact!is!that!the!people!in!this!field!are!studying!different!types!of!bacteria,!

Cas9 is a highly programmable nuclease tool for modifying DNA sequences in the genomes

of various organisms. Directed by sgRNAs with a 20-nt DNA binding sequence, Cas9-induced and! they! discovered! that! different! species! of! bacteria! are! able! to! produce! different! CasSlike!

! - Efficient in

sequence-specific DNA DSBs have been used to introduce nonhomologous end joining (NHEJ)– system.!

Transcription'repression'mediated'by'Cas9'

mediated sequence-specific insertion or deletion (indel) mutations in human endogenous genomic

loci, which often lead to loss of function of target genes (17, 42, 68). The CRISPR/Cas9 system In! the! last! few! months,! a! Cas9Slike! protein! called! cpf1! has! been! discovered,! and! it! has! with wt Ca

is highly programmable and multiplexable. When introducing multiple sgRNAs, it can simulta-

neously edit several sites within a mammalian genome (17, 69) and can generate animals that carry completely!different!requirement,!more!specific!respect!to!Cas9.!

mutations in multiple genes (40, 55, 111). Multiple DSBs simultaneously induced by Cas9 and

multiple sgRNAs can promote large or small chromosomal rearrangements between these DSBs, !

including interchromosomal translocations and intrachromosomal inversions, and could therefore Engineering'of'cas9'

serve as a potential tool for the study of genomic rearrangements (14, 122). The rearrangements - More restr

and inversions likely occur through the NHEJ pathway of DSB repair, as they involve the joining Other!systems!that!people!are!now!trying!to!use!is!modify!the!cas9.!!!

2a).

of mismatched ends (Figure

Besides NHEJ, DSBs introduced by CRISPR can also trigger DNA repair through homology- site

This!is!because!one!problem!concerning!the!Cas9!is!the!length!of!this!protein!that!is!codified!

!

directed repair (HDR). In the presence of a single-stranded oligonucleotide or double-stranded

plasmid DNA donor template, HDR can mediate the precise replacement or insertion of DNA

The!dCas9!can!work!also!as!a!roadblock,!because!it!blocks!the!transcription!and!the!presence! by! 1300! nt,! so! it! is! difficult! to! fit! this! gene! for! the! application! with! adenoviruses,! it! is! not!

2a).

sequences from the template (Figure This allows precise gene modifications such as coding

of!dCas9!in!this!way!it!can!induce!the!release!of!RNA!polymerase.! compatible!because!it!is!too!large.!It!is!also!borderline!for!lentivirus.!!

sequence replacements, including but not limited to targeted mutagenesis, gene correction, and

However,! we! can! also! obtain! more! stringent! and! effective! transcription! repression! creating! People!are!now!trying!to!obtain!“nano*Cas*9”!or!to!identify!other!enzyme!that!has!likely!cas9!

www.annualreviews.org Studying Genomics and Diseases with CRISPR/Cas9 14.3

chimera!able!to!modify!this!locus!from!the!epigenetic!point!of!view.! activity!with!other!sequences.!!

* !

* Different'Cas'(slide75)'

Current'pitfall'of'CRISPR?Cas9' Different!Cas9!are!reported!coming!from!different!bacteria.!!

The!biggest!problem!of!the!CRISPR!system!are!the'off'target'effects.!Indeed,!it!is!true!that!20!nt! The! first! line! is! the! canonical! Cas9,! but! there! are! other! cas9! exploiting! different! PAM!

are!enough!to!confer!the!specificity!of!almost!one!position!into!the!genome,!but!unfortunately! sequences! (also! longer! PAM! sequences),! and! this! can! be! another! method! to! enhance! the!

the!Cas9!is!also!able!to!induce!the!cleavage!in!other!positions!very!similar!to!the!target!one.! specificity,!because!the!sequence!that!is!required!to!target!is!longer.!The!problem!is!that!the!

This!can!be!be!a!very!serious!problem!when!we’re!performing!gene!editing!in!which!we!have! space! of! mutation! is! lower,! because! the! target! that! can! be! treated! with! the! CasS9! are! in! a!

to!consider!all!the!variants.!! smaller!number!in!the!genome.!

Moreover!Cas9!recognition!is!mainly!rely!on!about!15bp!upstream!of!PAM! Ascp1*is!completely!different!from!other:!the!PAM!requires!simply!three!T.!It!is!cutting!not!in!

proximity! to! the! PAM,! but! it! cut! 24! nt! downstream! to! the! PAM! sequence.! Ascp1! is! able! to!

induce!a!protruding!end!(not!a!blunt!end),!and!the!distance!between!the!PAM!and!the!ends!is!

really!high.!!

The!fact!of!having!protruding!end!is!important!for!the!application!in!which!we!want!to!have!a!

! homology!recombination.!!

How'to'avoid'off?target'effects' !

To!prevent!this!situation!we!can!optimize!all!the!design!of!the!different!GuideSRNAs.!! !

Another!important!thing!is!to!have!a!CRISPRSCas9!system!that!is!able!to!works!only!for!a!little! !

period!of!time.!! !

To!achieve!this!we!can!use!a!Cas9'that'is'bleachable!for!example.!! !

! 53! ! 54!

Delivery*of*cas9* 15.2.2017!

Concerning!the!delivery!of!Cas9,!more!efficient!delivery!method!would!be!crucial!for!in!vivo! Single*cell*analysis*

application.! The!main!reason!to!use!it!was!that!scRNASseq!is!the!lack!of!biological!material.!

In! mouse,! we! can! use! different! approaches;! one! of! the! most! interesting! approaches! is! the! !

presence!of!a!couple!of!strain!in!mouse:! Why!single!cell!analysis?!

! In!one!case,!we!have!the!constitutive!expression!of!Cas9!in!all!the!cells!and!we!have!not! We!can!consider!the!colour!code!as!a!gene!expression.!!

to! deliver! the! Cas9,! we! simply! have! to! deliver' the' vector' with' the' guide' RNA' and' On! the! right! the! two! groups! of! cells! have! a! very! different!

eventually'the'homology'sequence'of'DNA'for'HR.' expression! of! genes,! but! if! we! sequence! with! the! canonical!

! Another!interesting!trick!for!other!organisms!and!for!having!a!safer!genome!editing!is! RNASSeq!we!obtain!the!as!result!the!media!between!the!two!

the! delivery' of' the' ribonucleoprotein' complex.! It! is! possible! to! produce! the! Cas9! in! populations.!So!in!a!population!average!it!is!not!possible!to!

bacteria,!purify!this!enzyme!and!couple!it!with!the!GuideSRNA!simply!mixing!them!in! distinguish! between! a! state! in! which! all! cells! have! an!

solution.!! intermediate!phenotype!(pink)!and!one!in!which!half!are!on!

This! ribonucleoprotein! complex! can! be! then! transfected! into! cells.! We! are! basically! (red)!and!half!are!off!(white).!

performing! a! pals! of! gene! editing,! because! we! are! introducing! into! the! cells! a! fully! !!

functional! Cas9! containing! the! GuideSRNA;! this! Cas9! is! able! to! induce! the! genome! So!why!single!cells!analysis?!

editing,!and!then!it!will!be!destroyed!after!few!hour.!! S A!population!of!cells!shows!a'strong'heterogeneity!–!every!cell!is!different!–!or!not!all!

This! is! safer! way! because! we! are! not! introducing! neither! a! virus,! nor! a! DNA! that! is! the!cells!are!in!the!same!phase!of!the!process!and!therefore!show'different'signals!

continually!producing!the!Cas9.!! S With!the!greater!part!of!current!experimental!tools!the'average'signal'of'the'majority'of'

We!can!now!use!CD4!cells!from!patients,!perform!in!it!the!k.o.!and!then!reinject!them.!! population' may' mask' the' signal' of' the' minority,! or! certain! dynamic! behaviour! like!

* oscillations!is!not!visualized!on!a!cell!population!level!but!can!be!seen!with!singleScell!

Useful'resources' resolution!

! Addgene! →! reservoir! of! plasmid! and! reagent.! It! is! possible! to! buy! a! plasmid! through! !

this!resource.! This!brings!us!to!some!biological!questions:!!

! Toolgen!! 1. What!cell!types!exist!within!a!population!of!cells!in!a!particular!sample?!

! 2. What!does!this!highSresolution!view!of!cellular!transitions!tell!us!about!switches!in!cell!

Correction'of'mutation'in'zygote'states'of'human' state?!!

About!modification!in!humans:!it!is!possible!to!modify!the!monkeys,!but!we!have!really!more! 3. How! can! geneSregulatory! networks! be! constructed! using! geneStoSgene! expression!

knowledge!and!techniques!about!the!treatment!of!the!human!embryo!respect!to!the!monkey.! correlations!and!clustering!of!genes?!

! !

Assisted'reproduction'technology'is'common'' !

In!the!USA,!more!than!65!thousands!infants!were!obtained!by!in!vitroSfertilization,!so!1%!of! Identification'of'cell'types'population'

all!the!infants!derive!from!assistant!preSreproduction!technology.!!

We! can! introduce! the! sperm! into! the! egg! and! the! reinsert! it! in! the! blastocyst! then! into! the!

uterus.!

Another! important! issue! it! that! now,! it! is! feasible! to! perform! oneStranscriptome! analysis!

starting!from!one!cells,!because!we!can!remove!one!cell!in!this!stage!without!any!problem.!!

We!can!perform!the!treatment!of!the!zygote!with!CRISPRSCas9,!wait!for!the!8Scell!state,!then!

remove!one!cell,!perform!the!analysis!and!then!decide!the!embryo!right!to!be!implanted.!! !!!!!!!!!!!!!!!! !

This!approach!therefore!put!the!basis!for!“geneticallySmodified!humans”.!This!application!can! PCA!can!be!used!to!identify!new!cells!types!in!a!population.!There!are!many!informatics!tools!

brought!many!important!changes,!but!it!is!necessary!to!consider!also!the!ethical!implications.! that! enable! you! to! work! with! set! data! and! with! cluster! cells! that! are! similar! from! the!

! transcriptional!profile.!

The! Beijing' genome' institute! is! the! most! important! institute! of! genome! sequencing! in! the! Once!isolated!the!different!type!of!cell,!we!can!investigate!each!subSpopulation!analysing!the!

world,! it! has! invested! a! lot! of! money! for! the! screening! before! embryo! stage.! They! are! also! genes!expressed!in!these!cells.!You!can!find:!

performing!the!project!concerning!the!sequencing!of!a!million!people.!! S New'phenotypic'markers'

In! few! years,! we! will! probably! have! the! possibility! to! know! which! are! the! genes! and! the! S Selectively!isolate'the'gene'expressed'in'one'specific'population!→!looking!for!the!genes!

different! alleles! associated! with! different! condition,! and! this! can! lead! to! the! prevention! of! that!are!highly!expressed!in!one!single!population!

pathologies,!but!can!also!lead!to!a!dangerous!field,!like!the!possibility!to!have!normal!babies! S Calculate!the!frequency'of'your'population!→!useful!in!cancer!in!which!is!important!to!

or!designed!babies,!and!this!would!lead!to!important!implications!for!our!society.!! analyse!the!%!in!the!original!sample!

The!problem!is!that!no!one!is!discussing!these!issues.!! S Study!the!transition'between'one'population'to'another!→!studying!the!gene!expression!

! during!the!two!phases.!

! 55! ! 56!

! Single*cell*DNA=Seq*

There!are!many!techniques!to!isolate!and!perform!singleScell!DNASSeq:!

! Micromanipulation!→!

! Laser'capture'microdissection'→!very!difficult!because!it!is!a!long!process!

! FACS!!

!

! What! mainly! changed! is! the! time! required! for! the! process,! thanks! to! two! techniques:!

! microdroplets' and' microfluidics.! These! two! techniques! are! also! more! efficient! because! they!

! enable!to!set!up!the!volume!of!the!reaction!in!a!little!volume,!so!increasing!the!efficiency!of!the!

Single!cells!sequencing!follows!the!same!protocol!for!DNA!or!RNA:!! reaction.!

S Create!a!single?cell'suspension!! !

S Cut!the!single!cell!and!capture'DNA'or'RNA! The! crucial! step! of! DNASseq! is! the! amplification.! There! are! two! different! types! of!

S DNA/RNA' amplification! the! amount! of! DNA/RNA! is! a!

→! amplification!protocols:!

limiting'factor!for!the!sensitivity!of!this!technique,!so!we!have! Multiple*Displacement*Amplification*(MDA)!!

to!introduce!an!amplification!step! It! uses! a! DNA! polymerase! with! a! strand! displacement! activity.!

! Following! a! random! priming,! DNA! is! synthesized! in! the! 5’! 3’!

→!

! direction.!!

! When!the!3’!end!of!an!extending!string!of!nucleotides!reaches!the!

! 5’! end! of! the! adjacently! primed! string,! it! displaces! the! latter!

! resulting! in! new' single' strand' DNA' template' for' displacement'

! amplification.' This! is! a! quite! stable! interaction! and! we! can!

! synthesize!very!long!fragments.!

! The!advantage!is!that!you!don’t'have'multiple'runs!like!PCR!but!is!

! just!one!reaction;!moreover!this!DNASpolymerase!has!a!very!high!

! proof! reading! activity! so! the! possibility! to! introduce! errors! in!

Sample'preparation'workflow' amplification!it!is!really!low,!and!since!with!this!method!you!have!

just!one!cycle,!we!have!fewer!errors!than!the!other!one.!

!

PCR*method*(PEP=PCR)!!

• Based! on! randomization! of! primers! and! continuous! cycles! of!

PCR.!The!disadvantage!is!that!the!probability!to!introduce!errors!

! is!higher.!

The!sample!preparation!occurs!in!three!steps:! Why!are!there!so!many!copies!of!primers?!

1. Dissociation! you! can! use! different! methods! (enzymatic,! chemical,! mechanical);! all!

→! Because! random! primers! are! random! so! you! don’t! know! the!

the!conditions!have!to!be!set!up!depending!on!the!tissue! sequence:! they! can! bind! everywhere! in! the! genome.! Different!

2. Enrichment!→!you!can!use!specific'markers!expressed!on!the!cell!of!interest!to!enrich! primers! can! bind! different! zones! of! your! strands! so! the!

them;!you!mark!them!with!labelled!Ab!and!you!analyse!the!cells!with!FACS!analysis!so! polymerase!transcribes!really!long!fragments.!!

that!you!can!enrich!the!suspension!with!the!cells!that!express!this!marker! Using! MDA! protocol! each! transcription! is! independent! and! you!

! don’t! have! PCR.! Using! the! PCR,! the! probability! to! introduce!

errors!is!higher.!!

!

!

!

Applications!

! !

3. Quality'control!→!you!need!vital*cells!in!order!to!perform!the!analysis;!it!is!a!check!of! Cancer!

vitality!and!we!can!also!calculate!the!diameter!of!the!cell.! Cancers! are! very! heterogeneous! populations:! in! particular! some! cells! can! be! considered! as!

! drivers! in! which! the! mutation! can! confer! a! clonal! advantage! and! positively! select! the! cell!

! during!the!evolution!of!cancer.!

!

! 57! ! 58!

The! progressive! accumulation! of! somatic! mutations! results! in! a! heterogeneous! ! polyclonal! The! protocol! currently! used,! was! created! for!

tumor,!in!which!different'clones'may'differently'respond'to'treatment.! genomic!DNA!and!it!has!been!adapted!to!single!cell!

! cDNA.!It!is!a!trasposase'mediated'protocol'(Nextera!

So,!if!you!have!a!polyclonal!tumor,!different'drugs'can'target'different'clones!and!you!have!to! protocol)! in! which! you! incubate' cDNA' with'

select! clones! in! order! to! see! which! is! the! most! aggressive! to! treatment.! Using! single! cell! trasposomes' that' are' able' to' fragment! your! DNA!

analysis!we!can!resolve!intraStumor!cell!heterogeneity.!! and! insert! specific! tasks' that' are' known' sequences!

! (the!blue!one!and!the!green!one).!!

Cancer! develops! through! a! multistep' process! in! which! normal! cells! progress! to! highly! Once!you!have!fragmented!DNA!in!2!fragments!you!

malignant!tumors!via!repeated!cycles!of!clonal!expansions.!DNASseq!promises!to!address!key! can! use! these! 2! sequences! to! amplify! the! library!

issues!in!cancer!research,!including!resolving!intraStumor!heterogeneity,!tracing!cell!lineages,! and!to!insert!specific'barcodes'that'are'able'to'label'your'cells.!Barcodes!are!known'DNA'

understanding!rare!tumor!cell!populations!and!measuring!mutation!rates.! sequences'inserted'in'each'sample!useful!to!associate!the!reads!to!cell.!

This!is!important!because!you!can'target'selectively'the'most'aggressive'clones.!! !

! Cel=seq!→!it!is!similar!to!in!vitro!transcription,!so!you!don’t'

Metagenomics! have' PCR' amplification.! You! have! an! oligodT' primer! used! as!

Our! understanding! of! bacteria! is! very! limited! to! those! few! species! that! can! be! grown! in! RTSprimer!which!binds!the!polySA!on!mRNA!and!you!have!a!

culture.!! barcode!into!the!primer;!then!you!have!a!promoter!for!RNAS

The! study! of! single! cells! provides! a! very! accurate! resolution! of! microbial! population! by! the! polymerase.! Once! you! synthesize! cDNA,! the! polymerase! is!

identification!of!lowSabundance!species.! able!to!actively!transcribe!RNA.!!

Single! cell! sequencing! is! the! best! approach! to! charachterize! organisms! that! are! difficult! to! You!can!use!this!RNA!to!generate!libraries.!!

culture!in'vitro.! !

All!the!previous!analysis!of!bacteria!usually!purified!them!and!sequenced!their!16S!rRNA,!to! !

identify!mutations!derived!from!this!sequence!in!order!to!create!a!genomic!tree.!! !

But!all!these!analyses!were!performed!in!bulk,!but!the!problem!is!that!not!for!all!bacteria!is! !

present!a!reference!genome!to!which!you!can!map!their!genome.! Embryogenesis!

This!approach!allows!you!to!generate!reference!genome!for!species!that!are!not!sequenced! Embryonic!development!can!be!considered!like!the!transition!of!differentiation!from!the!

before.!It!gives!you!information!about:!philogeny,!community!composition,!genes!or!pathways! single!cell!to!the!wholeSorganism!level.!

and!strain!variations.! Measuring!the!gene!expression!in!individual!cells!is!crucial!to!understand!the!gene!regulatory!

One!of!the!major!fields!is!microbiota!study.! network!controlling!human!embryonic!development.!!

! You! can! select! the! genes! that! are! activated! in! each! different! stage,! and! this! allows! you! to!

! isolate!the!genes!responsible!for!the!transition!from!one!phase!to!the!other.!

Single*cell*RNA=seq* !

!

The!second!part!of!the!flow!chart!is!related!to!RNASseq;!for!this!technique!we!have!the!same! Unbiased'sampling'and'transcriptome'profiling'of'single'cells'

problems!and!almost!the!same!steps!of!DNASseq:! One!of!the!main!fields!of!application!is!unbiased!sampling!which!is!the!most!useful!technique!

1. Dissociation!and!cell!suspension!production! to!sequence!single!cells.!It!is!used!when!you!want!to!analyze!tissues!and!you!expect!to!obtain!

2. Capture!single!cells!and!collect!RNA! your!population!among!unknown!cell!populations.!!

3. Amplification!step!! For!example:!a!sample!of!cells!is!taken!from!the!tissue!of!interest,!with!the!aim!of!obtaining!a!

! representative!sample!of!the!types!of!cells!that!are!present!in!the!tissue.!!

Amplification'protocols' Each!cell!is!profiled!using!single!cells!RNA!seq!→!resulting!expression!profiles!are!clustered!

In!order!to!amplify!RNA!we!can!use!two!protocols:! together,! so,! basically! we! reconstruct! the! tissue! of! origin! from! the! RNASseq! data.! Nobody!

Smart=seq! it! is! a! common! reverse! transcription:! you! have!

• →! knows!if!there!are!some!populations!that!are!not!known!yet.!!

mRNAs!with!polySA!tail!and!you!use!oligo?dT'primers!to!prime!the! !

RTSreaction.! !

In!this!case!the!reverse!transcriptase!adds!at!the!end!a!poly=C*tail! !

and! that! can! be! used! to! anneal! another! primer! with! specific! !

sequence!at!the!3’!and!5’!ends.! !

These!specific!sequences'are'known:!so!we!can!use!them!to!amplify' !

all'the'cDNA!generated.!! !

After!having!generated!the!cDNA!and!amplified!the!fragments,!all! !

the!transcripts!are!represented!in!your!sample!and!you!can!go!on! !

generating!the!library.!!

! 59! ! 60!

Platforms'to'perform'RNA?seq' 7. cDNA' quantification' and' library' preparation→! generate! the! library! with! Nextera!

protocol!(transposome!with!adapters!combined!with!template!DNA)!and!fragment!the!

* cDNA.!Each!barcode!is!specific!for!your!cell!and!then!we!can!sequence!them.!

Fluidigm*C1*system!! 8. Sequencing'!

This! machine! is! used! for:! cell! preparation,! cell! isolation,! supporting! RTSPCR! and! next! !

generation!sequences!techniques,!discovery,!validation!and!screening.!

Can!support!some!protocols:! Movie:!

! Gene!expression! ! Cells'are'loaded'in'the'chip!and!flow!one!by!one!inside!it!until!they!are!captured!in!sites!

! microRNA!gene!regulation! ! Once!cells!are!captured,!the!other!cells!goes!on!flowing!until!all!the!96!tracks!are!full!!

! DNASseq!genome!sequencing! ! Then!reagents'are'injected'in'the'chip,!so!the!cells!are!focused!in!this!area!and!reagents!

! mRNA?seq'transcriptome'profiling'' flow'through'the'capture'sites.!!

! ! The!cells'are'lysed!and!the!content'flows'to'the'first'chamber!!

This! machine! uses! disposal' chips! in! which! cells! are! loaded! and! automatic' cell' lysis! is! ! cDNA' synthesis' occurs! RT?reagents' are' injected! in! the! chip! and! RTSreaction! is!

→!

performed;! so! you! don’t! have! nucleic! acid! extraction! because! an! automated' reverse' performed!

transcription'occurs'inside'the'chip.!In!this!chip!we!can!capture!until!96'cells!from!suspended! ! At!the!end!the!cDNA'is'amplified'in'the'last'chamber!

cells!in!the!sample!and!reverse!transcribe!everything!in!a!nanoliter'volume.!! ! The!protocol!takes!almost!6h!depends!on!the!cells.!!

At!the!end!of!the!protocol!from!the!chip!we!can!collect'cDNA'from'96'different'cells,!transfer!it! ! Then! the! cDNA'is'pushed'in'the'lateral'wells! and! you'can'collect'it! and! it! is! use! it! for!

in!a!plate!and!use!this!cDNA!for!RT?PCR'experiment'or'RNAseq'analysis.! further!experiment!(PCR!or!RNA!seq…)!!

Single'cell'mRNA'sequencing'protocol?workflow' !

Step!by!step:! !

1. Dissociate'the'tissue!to!have!a!cell!suspension! Chromium*10X*Platform**

2. Select'cells!according!to!their!size!→!you!have!to!check!cells!dimensions!because!you! It!is!a!dropSbased!system!for!mRNA!seq!and!that!allows!you!to!sequence!up!to!ten!thousands!

have!to!use!different!chips!according!to!the!size.!! cells!for!sample.!It!is!very!fast!protocol!(10’)!and!this!is!an!important!feature!because!you!can!

3. Load'and'capture'cells!→!there!are!different!types!of!cell!capture!that!are!engineered! minimize' the' stress! in! your! cell:! having! no! stressed! cells! it’s! important! because! stress! may!

specifically! for:! small! cells! (5S10µm)! medium! cells! (10S17µm)! and! large! cells! (17S affect!also!final!results!(increase!in!apoptotic!genes!or!stress!related!genes).!

25µm).!!In!each!chip!is!present!a!microfluidic'circuit!in!which!you!inject!cells!in!wells! !

(are! all! around! the! microfluidic! circuit).! From! these! wells,! injected! cells! can! pass! How!it!works:!

through!patterns!in!which!can!run!into!some!captureSsites!where!they!are!captured!in! You!use!a!suspension!of!beads!(which!are!synthetic)!conjugated!to!specific!oligonucleotides.!

according'to'their'size.!In!each!captureSsite!there!is!also!a!sequence'reaction'chamber!in! These!oligonucleotides!contain:!

which! all! the! reactions! happen.! In! this! chip! you! can! also! inject! reagents! that! you! are! ! Poly?T!→!used!for!RT!reaction!(as!the!others!protocols)!!

going!to!use!to!generate!RT!amplification.! ! 10xBC!→!a!special!barcode!that!is!different!for!each!bead.!!

So,!you!have!a!suspension!of!beads!containing!more!than!700’000!different!barcodes!and!you!

can!associate!one*bead*to*one*cell!(so!one!barcode!to!one!cell)!mixing!these!beads!inside!the!

chip.!!

Chip!is!again!a!micro'fluidic'platform!in!which!you!inject:!

! Beads!

! Cells!suspension!!!

! ! Enzymes!to!perform!RT!reaction!

4. Wash' and' stain! calcein! homodimer! stain! is! a! specific! stain! that! allows! you! to!

→! ! Oil!

discriminate!between!alive!and!dead!cells:!green!cells!are!alive!and!red!cells!are!dead! !

5. Lyse'cell! The! passage! of! these! reagents! through! the! oil! flow!

6. RT' reaction' with' the' Smart?seq' protocol! it! is! a! RT!

→! generates!some!drops!like!small!bubbles.!!

transcription! with! amplification! of! cDNA.! At! the! end! of! the! Every'drop'contains'a'bead'with'the'specific'barcode'

procedure! you! can! collect! from! lateral! wells! of! the! chip! 96! and' a' cell.! Each! drop! is! a! sort! of! micro' chamber'

cDNAs! and! you! can! check! the! concentration.! You! need! a! reaction!so,!all!the!reactions!occur!in!these!drops.!!

specific! concentration! for! sequencing! protocol.! You! can! Then! you! collect! these! drops! and! perform! a! RT'

perform! cDNA! electrophoresis! and! this! is! the! big! reaction!inside!them!and!then!remove!the!oil.!

corresponding! to! the! cDNA.! The! amount! of! cDNA! you! can! Then! you! can! pool! all! the! generated! cDNA! because!

obtain!is!really!different!and!depends!on!the!cells!type!so;!if! they! are! barcoded! so! each! cell! has! a! specific!

you!are!using!large!cells!you!will!have!more'RNA!than!using! barcode.!!

small!cells. !

! !

! 61! ! 62!

CHROMIUM'10X!Protocol' Data*Analysis*methodologies*

The!grey!dot!is!the!bead,!while!in!green!there!is!the!oligo!conjugated!to!the!bead.!! The! single! cell! RNASseq! allows! us! to! perform! the! transcriptome! of! a! single! cell! and! it! is! a!

The!oligo!has!a!dT'region!that!anneals!the!polyA!of!the!mRNA!and!that!is!used!for!the!RT! powerful!tool!to!discover!the!variability!profile!cellStoScell!from!a!genomic!point!of!view.!

reaction.!! Machine!learning!algorithms!are!used!to!find!hidden!structure!in!unlabelled!data,!so!that!you!

First! strand! is! synthesized:! the! reverse! transcriptase! adds! a! polySC! tale! as! in! SmartSseq! can!cluster!different!cells!→!capture!the!meaning!of!these!cells.!

protocol!(but!here!5’!is!lost);!after!template!switching!polySC!tale!is!used!to!amplify!cDNA'in! The!starting!point!is!a!huge!matrix!in!which:!

the! preSamplification! step! so,! you! use! these! primers! to! amplify! what! you! have! reverse! Columns*→!the!number!of!single!cell!samples*

transcribed.' Rows!→!all!the!genes!that!are!detected.!!

The! first! step! is! the! alignment! of! the! reads! to! the! human! genome! and! the! generation! of! the!

counts,!it!is!the!same!procedure!as!the!RNASseq!bulk.!We!can!generate!the!counts.!

The! second! analysis! is! quality! control,! and! then! we! have! a! lot! of! difference! procedures! to!

reduce!the!data:!

! Principal*Component*Analysis*(PCA)!

We!start!from!the!matrix!and!we!recapitulate!the!information!of!the!gene!expression!in!a!2D'

! plot.!!

PCA! is! a! statistical! procedure! that! uses! an! orthogonal! transformation! to! convert! a! set! of!

observations! of! possibly! correlated! variables! into! a! set! of! values! of! linearly! uncorrelated!

variables!called!principal'components.!

The!PCA!transforms!the!big!matrix!in!a!new!system!of!two!coordinates:!

Principal'component'1! first! coordinate;! it! is! the! projection! of! the! data! in! a! new!

o →!

dimension!that!captures!the!high'variants'between'all'the'data'

! Principal'component'2! coordinate;! we! have! the! remain! variants! that! are!

o

When!you!generate!the!library,!all!fragments!have!a!specific'barcode'associated'to'the'cell!but! →second!

orthogonal!to!the!PC1.!

if! you! pool! more! samples! together! you! need! also! a! barcode! to! associate! each! cell! with! its! Each!single!cell!is!a!point!in!this!2D!plot:!so!near!points!have!a!similar!gene!expression!profile.!!

sample.!!

So!if!you!load!4!samples,!the!sample!A!should!have!a!sample'barcode!to!distinguish!it!from!the! !

B,C!and!D!samples!but!you!also!have!to!be!able!to!distinguish!the!different!cells!between!the!A!

sample.!!

At!the!end!you!have!these!kinds!of!fragments!in!which!the!sample*index!identifies!the!sample!

and!the!10x!barcode!identifies!the!single!cell.!! ! ! !

! We! can! recapitulate! the! cellular! diversity! with! this! 2D! plot! that! describes! the! variability!

Disadvantages'and'advantages'of'Fluidigm'or'Chromium10x'' within! the! data,! and! this! allows! us! to' detect' the' outlier' samples! which! can! be! filtered! and!

The!main!difference!between!the!two!protocols!is!the'information'you'can'get.'' visualized!in!the!smaller!subsets.!!

Fluidigm!is!more!labor!intensive!compared!with!the!other!one!but!you!are!able!to!sequence!all! We!can!also!visualize!the!relationship!between!the!variables,!depending!on!their!proximity:!if!

the! mRNA! until! the! 5’end.! This! gives! you! many'more'information! compared! to! the! 10X! that! the!variables!are!close!to!each!other!are!similar!in!their!expression!profile.!!

sequences!just!the!3’!of!the!mRNA,!especially!about!isoforms!and!splicing.!! This!is!why!it!is!called!data!reduction,!because!you!start!from!a!huge!matrix!and!obtain!less!

But! the! advantage! of! 10x' is' that' it' is' really' high?throughput! in! a! single! chip! now! you! can! data.!

sequence!80000!cells!for!chip.!! !

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t=SNE!(t?Distributed'Stochastic'Neighbor'Embedding)! We! can! also! perform! this! analysis! at! the! gene! level! (and! not! at! the! sample! level):! you! can!

It!is!another!approach!of!data!reduction!that!is!more!powerful!because!it!also!captures'distinct' detect!genes!highly!expressed!in!one!cluster!and!genes!highly!express!in!the!other!cluster.!So!

sub?clusters' of' cells! compared! to! the! PCA.! So! we! can! first! perform! a! PCA! for! the! higher! it!is!possible!to!identify'groups'of'gene'that'are'co?regulated!(while!at!the!sample!level!we!can!

variants,!then!we!can!perform!a!tSSNE!to!capture!the!subclasses!of!clusters.!So!it!is!possible! identify!classes!of!tumors).!

also!to!detect!the!subSclusters!of!cells!that!express!a!different!characteristic!marker! !

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!PCA!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!tSSNE! This!is!a!Heat*map:!

In!the!column!there!are!samples!and!in!the!rows!there!are!genes.!!

Color! code! is! related! to! the! expression' of' these' genes:! green! means! low!

expression!while!red!means!high!expression.!!

In!this!case,!if!we!performed!analysis!at!sample'levels!you!can!find!2!different!

clusters:!the!blue!one!and!the!orange!one.!

Performing!cluster!analysis!at!gene!level!you!can!detect:!

! Genes!that!are!high!or!low!expressed!in!blue!cluster!!

! Genes!that!are!high!or!low!expressed!in!the!orange!cluster!!

So! with! the! clustering! analysis! at! the! sample' level! it! is! possible! to! identify!

!!!!!!!!!!!!!!!!!!!!!!! ! cluster!of!tumors!or!cell!type!that!have!a!similar'expression'profile.!

* At!the!gene'level!you!can!identify!groups!of!genes!that!are!coregulated.!!

The$PCA$plot$is$less$effective$at$separating$cells$into$many$different$clusters.$! !

This%is%because%the%first%two$principal$components$are$driven$by$strong$differences$between$ !

specific' subpopulations,' which' reduces' the' resolution' of' more' subtle' differences' between' !

some%of%the%other%subpopulations! !

* We!have!a!lot!of!technical'confounding'factors!in!single!cell!analysis,!because!we!analyse!a!lot!

* of!cells!and!genes.!For!this!reason!we!use!the!spike=in!molecules:!synthetic'sequences'added'to'

Cluster*analysis* the'mix'in'a'constant'volume,!so!in!each!cell!we!will!have!the!same!quantification!of!the!spikeS

It!is!a!group!of!analysis!that!group'cells'according'to'the'similarity'in'the'expression'profile:!if! in!transcript.!

we! have! the! similarity! of! cells,! they! are! close! to! each! other! and! we! can! detect! different! So! if! problems! occur! we! can! normalize! our! result! looking! at! spikeSins.! There! are! a! lot! of!

clusters!of!cells.!If!you!have!a!good!clustering!method!you!can!have!highSinterclass!similarity! technical!factors!that!could!lead!to!a!nonSequal!quantification!and!for!this!reason!is!important!

compared! to! the! other! clusters.! One! of! the! most! used! cluster! analysis! is! the! hierarchical' to!normalize'all'the'expression'values'of'all'genes'to'the'amount'of'the'spike?in.!!

analysis!that!starts!with!each!sample!that!is!a!separate!cluster,!then!tries!to!group!together!all! So!the!expression!of!the!spikeSin!can!be!used!to!compare!the!expression!of!the!genes!across!of!

the!samples!based!on!the!proximity.!! all!the!different!samples.!!

!

scRNA?seq'analysis'workflow'

The!workflow!is!made!up!by:!

1. Read!alignment!of!and!quantification!of!the!expression!values!!

It!is!quite!the!same!of!RNASseq!bulk.!So!we!perform:!the!trimming,!all!the!quality!controls!of!

the!RNASseq!data!and!we!obtain!the!matrix!of!counting!for!each!genes.!

! 2. Quality!control!

! We!have!a!lot!of!steps!of!quality!control!because!with!RNASseq!we!have!a!lot!of!things!related!

to!the!technical!noise.!We!want!to!discard!the!poor'quality'cells:!all!the!cells!with!insufficient!

number! of! genes! detected! and! all! the! cells! with! some! problems! from! the! technical! point! of!

This%produces%a%binary'tree'or!dendogram view!are!filtered!out!using!different!criteria.!

The"final"cluster"is"the"root"and"each"data"item" 1. %'evaluation'of'the'mapped'read'to'the'genome! cells! with! a! percentage! of! mapped!

is#the#leaf →!

read!lower!than!60%!are!filtered!out.!!!!!!!!!!!!

The$ height$ of$ the$bars$ indicate$ how$ close$ the$ 2. %'evaluation'of'ERCC'mapped'reads'→!ERCC!are!synthetic!sequences.!We!have!all!the!

items&are information! about! these! sequence! so,! parts! of! the! reads! are! related! to! ERCC! because!

are!sequence!that!are!normally!sequenced!during!the!sequencing!approach.!Given!that,!

part! of! the! reads! is! related! to! the! ERCC! and! the! other! reads! are! related! to! the! real!

!

! transcripts! that! mapped! to! the! human! genome! for! example.! If! the! percentage! of! the!

! reads!related!to!the!ERCC!is!very!high,!we!have!a!technical!problem,!so'we'discard'all'

! 65! ! 66!

cells'with'percentage'of'ERCC'higher'than'60%'because! it! means! that! the! information! 28.02.2017!

related!to!the!real!transcript!is!only!the!40%.!!!!!!! !

The*3D*genome*organization!

3. %!of'reads'mapped'to'the'mitochondrial'genome!→!cell!with!a!high!%!are!filtered!out,!!

because!mitochondrial!genome!is!an!indication!of!apoptosis.! Transcriptional!regulation!depends!on!the!3D!structure!of!the!genome.!!

4. Library'size! we! want! to! compare! cells! with! almost! the! same! library! size.! Library*

→! Epigenetics!is!something!that!is!on!the!genetics,!so!it!is!the!regulation!of!gene!expression!that!

size! is! total! sum! of! the! counts! across! all! features:! genes! and! the! spikeSin.! So' all' the' determine!cell!identity!and!its!dynamic.!

counts'related'to'the'genes'and'SPIK?IN'must'be'equal'for'all'the'cells.'! Our!idea!since!20S30!years!ago!was!that!the!DNA!corresponds!to!an!organism,!so!the!DNA!and!

5. Number'of'expressed'genes!→!cells'with'the'lower'number'of'detected'genes'are'filtered' the! sequences! are! very! important.! But! now! we! know! that! there! is! something! else! that! is!

out' because! probably! there! are! technical! problems! related! to! the! detection! of! the! epigenetics.!!

genes.!! If!you!look!at!homozygous!twins!and!have!exactly!the!same!DNA,!during!aging!there!will!be!

6. Perform'PCA!→!we!can!have!a!over!view!of!our!samples.!! some!kind!of!differences.!During!aging!differences!are!more!evident!because!the!organs!react!

! to!the!environment!in!a!different!way.!The!geneticists!are!studying!these!homozygous!twins!

3. Normalization* because! they! feel! the! environment! in! a! very! different! way! although! they! share! the! same!

Then! we! normalize! the! data:! all! the! confounding'factors'must'be'filtered'out! to! have! the! real! placenta.!!

expression!of!the!genes.!! We! have! different! cells! that! have! different! functions.! If! you! look! at! different! cells! they! are!

! phenotypically! different! from! skin,! from! neurons,! muscle! or! cardiac! cells.! They! also! have!

! different! functions,! because! the! cardiac! cells! and! muscle! cells! can! contract,! while! neurons!

scRNA?seq'applications' have! electricity! faculty.! They! are! very! different! one! from! each! other,! although! the! DNA! is!

exactly!the!same!because!we!use!this!DNA!in!different!ways.!This!is!the!epigenetic!regulation,!

One!of!the!main!goals!of!single!cell!RNASseq!is!the!identification'of'cell'type'and'cellular'state.! the!different!use!of!DNA.!!

When!we!have!the!PCA!we!can!perform!a!lot!of!downstream!analysis.!For!example!the!tSSNE! We! can! say! that! transcription'determines'the'cell'identity,! not! the! DNA! content! but! the! RNA!

detects!several!cluster!of!cells,!for!which!we!have!no!information,!and!we!want!to!add!a!sort!of! content.!During!the!differentiation,!starting!from!one!genome,!we!will!obtain!the!epigenome,!

label!on!this!cells.!How!can!we!add!a!label!and!see!that!this!cluster!is!CD4!cells!without!any! one!for!each!cell!type.!Moreover!during!the!differentiation!also!the!nuclear'structure'changes.!!

information?! Ex:!During!neural!differentiation,!you!start!from!this!nuclei!that!is!not!rounded!and!you!finish!

I!can!check!for!the!expression!of!a!specific!marker!and!so!I!can!assign!a!label!to!the!cluster.! with!the!rounded!nuclei;!so,!it's!not!only!the!RNA!content!that!is!changing!but!also!the!nuclear!

! structure.!!

It! is! also! possible! to! have! no! information! about! any! markers! and! for! this! reason! a! lot! of! !

statistical! approaches! are! now! developing! to! detect! the! genes! that! drive! the! different! By! definition! we! can! say! that! epigenetic' mechanisms' fix' in' time' and' space' specific'

variability! within! the! cluster.! These! genes! are! called! high* variable* genes:! they! are! very! transcriptional'programs:'

variable!genes!in!the!different!clusters,!so!specifically!expressed!in!a!cluster.!!

If!we!have!no!biological!information!we!can!detect!genes!that!are!specifically!expressed!in!a! Time!→!during!differentiation!we!have!a!constriction!of!the!transcription!and!there!are!

specific!cluster.!! also!different!factors!that!are!transcribed!

! Space!→!in!one!part!of!the!body!we!will!have!specific!cells!and!in!other!part!of!the!body!

Comparison!of!bulk'strategy!and!single'cells!from!bioinformatic!point!of!view!(final!slide):! we!will!have!different!cells!

In!quality!control!passage!there!are!more!steps!in!single!cell!analysis!because!we!have!a!lot!of! !

technical!noise!compared!to!RNA!seq.!BULK!analyses.!!!! Regulators! of! differentiation! process! are! Polycomb! proteins! that! are! transcription!

Normalization!passage!is!also!different!because!in!single!cell!RNAseq!analyses!again!we!have! repressors.!

much!technical!rumors.!! They! are! very! important! for! the! maintenance! of!

! the!cell!identity!and!for!all!kind!of!differentiation.!

! In! a! stem! cell! we! have! all' the' lineage' specific'

! genes' that' are' repressed' by' Polycomb' proteins.!

! They! sit' on' the' promoter! and! shut! off! the!

! transcription,! while! the! stem! cell! genes! are! on!

! because! they! have! to! maintain! the! proliferation,!

! the!stemness...!!

! When!differentiation!starts,!these!key!factors!can!

! jump! from' the' lineage' specific' genes' to' the' stem'

! cell' genes.! For! example! for! the! cell! type! A! you! need! the! expression! of! lineage! A! genes.! So!

! polycomb!will!leave!the!specific!promoter!of!the!lineage!A,!but!not!those!of!lineage!B!and!C,!

! and! it! will! repress! the! stem! cell! gene,! because! they! need! to! stop! proliferation! and! start!

! differentiation,! and! produce! all! the! proteins! that! are! specific! for! each! different! cell! type.!

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Another! aspect! of! genes! that! we! have! to! take! in! consideration,! only! 1.5! %! will! product! It! is! important! also! to! take! in! mind! that! you! don't! have! only'the'active'and'repressive'state:!

proteins.!We!were!used!to!think!that!proteins!make!the!function!of!the!cell,!but!this!is!not!true,! with! epigenetics! you! can! really' control' the' level' of' transcription! because! cells! needs! a!

because!you!have!the!non?coding'portion'that'regulates'the'coding'part'but'also'the'structure' precision!in!the!number!of!transcripts,!so!you!can!really!modulate!the!level!of!transcription.!!

of'the'cell.!! Example:! H3K9! methylation! is! associated! with! a! long! term! repression! and! heterochromatin!

The!genome!is!very!big!and!needs!to!be!packed!in!a!small!volume.!Even!during!the!cell!cycle! formation,! together! with! H3K27! methylation! that! is! a! short! term! repression! that! is! more!

this! condensation! is! different.! We! were! used! to! think! condensation! as! when! we! look! at! the! plastic.!

chromosomes! that! are! in! mitosis,! when! the! condensation! is! at! the! maximum! level,! while! !

during!interphase!the!DNA!is!not!condensed!in!this!way,!but!have!loop!and!specific!structures! Methylation*it’s!typical!of!long!term!repression!and!during!diseases!is!completely!altered,!for!

that!are!really!important.!You!need!to!condense!the!DNA!but!this!condensation'is'not'random! example!in!cancer.!

but!is!highly!regulated!and!differs!from!cell!to!cell.! !

DNA*HIGHER*ORDER*STRUCTURES:!

You! have! the! Histone! code,! the! methylation,! the! repressor! or! activator! and! it! is! a! monoS

dimensional! level,! and! then! you! have! the! biSdimensional! and! triSdimensional! type! of!

regulation.! BiSdimensional! regulation! of! transcription! →! for! example! chromatin! loop.! If! you!

have!a!regulator!and!a!promoter!that!are!flanking,!not!very!close!to!each!other,!then!you!need!

the!formation!of!the!loop.!You!can!also!have!the!loop!of!the!enhancer!on!the!promoter,!you!can!

also!sequester!the!enhancer!then!you!will!have!the!repression.!

!

Also!non*coding*RNA!can!regulate!transcription!in!different!ways!because!they!can!modulate!

!

! the!levels!of!RNA,!or!recruit!a!specific!factor!in!a!specific!place!on!the!chromatin.!

These!are!the!degrees!of!condensation;!when!we!think!at! !

the! DNA! we! think! at! a! mitotic! chromosome,! however! TADs!

this!state!of!the!chromosome!lasts!only!a!small!period!of! This! was! a! paper! published! in! 2005! in! which! the! authors! showed! that! if! you! stain! the!

its!life,!because!is!only!during!mitosis!that!our!DNA!has! chromosomes!with!different!colours!and!then!you!see!cells,!you!will!find!always!in!the!same!

this! aspect.! In! fact! only! during! the! mitosis! you! can! see! position!the!same!chromosomes.!Ex:!the!chromosome!8!that!is!apical,!will!be!apical!in!all!the!

condensed' chromosomes;! during! interphase,! that! is! the! cells!of!the!organism.!It!means!that!position'of'chromosomes'in'the'nucleus'is'conserved.!What!

most!of!its!life,!the!DNA!seems!to!be!completely!random,! was! really! interesting! is! that! not' only' in' one' organism' each' cell' has' the' same' chromosomal'

but! this! structure! is! highly'regulated.! In! interphase! you! position,'but'also'during'evolution'this'positioning'is'conserved.!!

have!certain!type!of!organization!and!during!the!mitosis! When! something! is! conserved! during! evolution,! this'means'that'

you!have!the!condensation!and!finally!you!can!separate!the!chromosomes.!! it' is' important' for' the' function.! The! places! occupied! by! the!

! chromosomes! are! called! Chromosomal* territories* (Cts)! and!

Starting! from! the! DNA,! this! is! an! overview! of! the! they! are! evolutionary! conserved.! This! doesn’t! mean! that!

different! levels! of! structure! that! gives! you! finally! chromosomes! are! fixed! in! the! nucleus,! because! they! are! very!

to!the!transcriptional!regulation.!! dynamic,!but!that!the!probability!to!find!that!chromosome!in!that!

On! the! DNA! we! have! the! histone' modifications' or' specific!region!is!really!high.!!

histone'variants!that!are!associated!with!a!specific! !

transcription! state;! then! there! are! also! the! Not!only!is!important!the!space!in!the!nucleus!that!is!occupied!by!the!chromosome!but!also!

chromatin'modifiers!that!are!specific!proteins!able! the!time'that'each'chromosome'spend'in'that'position.!!

to! read! the! histone! code,! DNA! higher! order! In!addition!we!have!to!consider!that!chromatin'presents'a'high'mobility,!so!you!continuously!

structures,! nonScoding! RNA,! TAD! (topological! have! this! movement! of! DNA! and! this! is! evident! in! an! undifferentiated! state! and! less! after!

associated! domains)! and! finally! nuclear! differentiation,!because!then!the!cell!is!specialized!and!have!to!produce!specific!proteins!that!

positioning.! are!typical!of!that!tissue.!!

! There!are!repressive'compartments,!where!you!can!find!genes!that!are!coSrepressed!and!active!

Histones! compartments!where!you!find!genes!that!are!coSexpressed.!!

They! have! different! tails! and! can! be! differently! modified;! the! combination! of! these!

modifications!gives!the!transcriptional!output.!It!is!very!important!to!notice!that!methylation!

of! histone! H3K4! gives! active! transcription! and! methylation! of! H3K9! gives! heterochromatin!

conformation.!!

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Moreover! we! have! the! compartimentalization! of! the! nuclear! processes:! if! you! stain! for! RNA!

pol! II,! the! enzyme! responsible! for! transcription,! you! will! obtain! a! particular! pattern! that!

represents!the!transcriptional'factories.!!

What!kind!of!dynamic!can!justify!this!kind!of!pattern?!Why!there!are!some!spots!that!have!a!

higher! amount! of! this! enzyme?! In! these! spots! all' the' enzymes' of' the' transcription' are' more'

concentrated'and'are'associated'the'genes'that'are'actively'expressed'in'that'cell.!!

You!don't!have!just!one!gene!but!each'transcription'factory'can'process'many'genes'at'the'same'

time.!So,!genes!that!need!to!be!coSexpressed,!even!though!they!were!very!far!from!each!other,!

are!find!together!in!the!same!transcriptional!factory.!!

! In!this!way!you!will!have!the!same!quantity!of!transcription!of!different!genes!that!maybe!are!

Very!recently!through!the!Hi?C'technology!was!determined!the!total!conformation!of!the!DNA.! in!the!same!pathway.!!

It!has!been!measured!the!frequencies!of!contacts!between!one!region!toward!other!regions,! It!is!not!the!polymerase!that!goes!to!the!DNA!to!make!the!transcription!but!is!the!DNA!that!

finally!you!can!have!an!entire!view!of!the!genome.!! goes!to!the!transcriptional!factor!to!be!transcribed.!!

It!has!been!showed!that!the!conformation'of'the'genome'is!not!random!but!is!highly!organized,! In!the!nucleus!we!have!also!replication'foci!and!the!DNA'repair'compartimentalization.!!

meaning! that! all' the' genomic' regions' that' share' the' same' level' of' expression,' were' found' !

together.!And!this!matches!perfectly!with!the!image!of!the!chromosome!territories.!! Nucleolus!

! It!is!a!space!in!the!nucleus!where!rDNA'from'different'chromosomes'are'transcribed,!not!all!the!

So! we! have! an! ordered! conformation! and,! depending! on! their! function! conformation! can! rDNA!are!transcribed!but!half!of!the!are!transcribed!and!half!of!them!are!repressed.!Again!you!

change.!The!3D'conformation'of'the'genome'is'very'important'for'the'function.!! have! a! compartimentalization! because! you! have! specific! parts! of! the! nucleoli! where!

Ex.! A! chromosome! in! which! you! have! several! genes! that! are! repressed! is! more! in! the! transcription! takes! place.! It's! very! important! to! study! also! the! nucleoli! because! in! diseases,!

periphery,!when!you!need!to!activate!one!gene!from!this!chromosome!it!protrudes!and!goes! such! as! in! cancer! you! have! an! increased! number! of! nucleoli:! it! means! that! there! are! some!

toward!the!centre'where'shares'the'active'components.!Chromosomes!not!fixed:!the!major!part! functions!that!are!altered.!!

of!the!chromosomes!stays!in!a!part!of!the!nuclei!but!they!can!reach!different!compartments! Also! during! reprogramming,! starting! from! a! differentiated! cell! going! back! to! the! iPS! state,!

when!there!is!activation!or!repression.!! from!many!different!nucleoli!finally!we!obtain!one!big!nucleolus!per!cell:!this!means!that!the!

! number!of!nucleoli!depends!on!cell!type!is!not!random.!

Nuclear*Structure! It!is!a!summary!of!the!environments!more!compatible!with!a!transcription!activation!and!he!

It!indicates!where!the!sequences!are!positioned!in!the!nucleus.!If!you!take!one!chromosome! others!that!are!compatible!with!transcription!repression.!

that!has!several!active!genes!stained!with!green!and!a!chromosome!that!is!poor!of!genes!or! !

that!are!repressed!stained!with!pink,!and!you!look!at!the!live!imaging,!you!will!see!always!the! So!the!active!compartments!are:!

chromosome!pink!at!the!periphery!and!the!green!ones!are!more!in!the!centre.!! Nucleolus' !transcription!of!rRNA'

We! understand! that! nuclear'periphery'is'more'compatible'with'repression'while'the'centre'of' Nuclear* pore'→!The!nuclear!pores!are!compatible!with!activation!even!if!they!are!at!

the'nucleus'is'more'compatible'with'the'transcription.!! the! nuclear! periphery,! that! is! usually! is! repressed.! Indeed,! near! nuclear! pores! are!

! localized!genes'that'need'to'be'transcribed'very'rapidly,!for!example!the!stress!genes:!in!

What'happens'during'diseases?!!While!in!a!regular!structure!with!active!domains!in!the!centre! this!case!when!you!have!an!activation!the!RNA'goes'directly'in'the'cytoplasm'and!then!

of!the!nucleus!and!repressive!domains!in!the!nuclear!periphery,!in!cancer!cells!everything!is! makes!the!protein.'

mixed! up:! you! will! find! repression! also! in! the! centre! of! the! nucleus,! accompanied! by! a! Transcriptional*factories'→!foci!in!which!are!localized!genes!that!are!coSregulated'

deformation! of! the! nuclear! membrane.! In! the! nucleus! we! have! different! structures! that! are! '

really!important!for!transcription!regulation.!! While!repressive!compartments!are:!

Nuclear* periphery! you! have! the! inner! nuclear! membrane,! at! the! inner! nuclear!

→!

• envelope!you!have!lamin!proteins.!Lamin'proteins!are!different!types,!they!are!close!to!

the!inner!nuclear!membrane!and!they!can!directly!bind!the!genomic!regions!which!are!

called!Lamin* Associated* Domains* (LADs):!they!are!big!genomic!regions!even!50Mb!

that!are!repressed.!At!the!border!of!LADs!you!find!specific'epigenetic'marks!such!as!CpG!

island,! CTGF! binding! site! and! also! histone! marks! H3K27me3! that! is! a! mark! of!

! Polycomb!important!for!the!cell!identity!

!

!

!

!

! 71! ! 72!

So,! what! is! interesting! from! the! epigenetic! point! of! view! is! that! the! same! mutation! can! give!

rise! in! a! family! (same! genetic! background)! only! to! the! muscle! phenotype! or! only! to! the!

cardiac!phenotype,!both,!or!even!can!be!asymptomatic.!So,!all!this!variability!is!independent!

from!genetic!and!probably!depends!on!the!epigenetic.!

!

! ! !

Nucleolus! at! the! edge! we! have! transcription'repression.! We! find! also! lamin! at! the!

→! So!we!can!have:!

• border!of!the!nucleolus:!these!are!called!Nucleolar*Associated*Domains*(NADs),!part! Monodimensional* regulation! of! the! transcription! contiguous! proteins! binding!

• →!

of!these!are!LADs!so!you!have!some!kind!of!shuffling!between!the!periphery!and!the! enhancers!and!promoters!enhancing!the!transcription!

border!of!nucleolus.!

Repression! of! transcription! at! the! border! of! nucleoli! is! needed! to! avoid! that! the!

polymerase! starts! to! transcript! and! doesn't! stop,! so! you! need! some! boundary' to'

localize' the' transcription' only' at' rDNA! level.! This! means! that! you! need! repression! !

immediately! out! of! the! cluster! of! rDNA,! otherwise! transcription! will! start! and! can! Bidimensional* regulation!of!the!transcription!→!flanking!and!distant!sequences!can!

transcribe!everything! control!gene!regulation!by!wrapping!or!looping!the!DNAs!!

Repressive*bodies!→!aggregation!of!proteins,!where!repression!takes!place:!the!gene!

• that! needs! to! be! repressed! can! localize! or! at! nuclear! periphery! or! at! the! edge! of! the!

nucleoli!or!in!Polycomb!bodies.!!

These! nucleolus! associated! domains! that! are! repressed,! are! H3K27me3' enriched! and!

they!share'some'sequences'with'LADs.!! !

! Tridimensional* regulation! genes! localized! on! distinct! chromosomes! can! be!

• →!

The! LADs! are! repressed! at! the! nuclear! periphery! and! enriched' of' heterochromatic' regions,! regulated!by!the!chromatin!environment!(euchromatin!and!heterochromatin)!

they!are!also!H3K9me3'enriched!(long!term!repression).!!

LADs'comprise'the'40%'of'the'genome:!so!there!are!some!LADs'that'are'common'for'every'cell!

type,! so! they! have! a! structural! role! and! function! as! anchor! points! to! organize! the! structure!

inside! the! nucleus.! Then! there! are! some! LADs' that' are' cell?specific! and! they! are! often!

associated' with' differentiation.! For! example,! the! lineageSspecific! genes! are! located! at! the!

nuclear! periphery! and! then,! when! differentiation! starts,! the! LADs! reSlocalize! toward! the! !

centre.! 4D*regulation!→!genes!localized!on!distinct!chromosomes!can!share!the!same!nuclear!

! compartment!in!the!same!moment,!so!also!the!time!in!which!interaction!takes!place!is!

Why'are'lamin'and'the'LADs'so'important'for'humans?!! important.!!

There!are!two!types!of!lamin:!

S Lamin! B! it! is! only! in! the! nuclear' periphery;! it!

→!

plays!a'structural'role!

S Lamin*A!→!it!is!also!located!in!the!nucleoplasm!and!

plays!a!regulatory'role.!! !

Lamin!proteins!regulate!several!nuclear!processes,!such!as! !

RNA!transcription,!DNA!replication,!nuclear!migration!and! Nuclear'dynamics'

being! involved! in! these! nuclear! processes,! in! fact,! The!nuclear!organization!takes!into!consideration:!

mutations! in! lamin! give! rise! to! human! disease! called! S Dynamic'flexibility!→!adaptable!response!to!the!environment!

laminopathies.! S High'level'of'the'order!→!functional!specialization!

! During! differentiation! the! loci! containing! upSregulated! genes! move! to! active! nuclear!

Lamin! B! mutation! is! not! compatible! with! life! because! it! compartments,! while! loci! containing! genes! that! must! be! repressed! move! to! other!

plays! a! structural! role! that! is! so! important! for! the! nuclei! compartments.!Of!course,!these!rules!are!not!universal!because!it!is!depending!on!the!specific!

and!when!upon!mutations,!cells!cannot!divide!and!survive.!! gene!and!it!can!change!from!cell!to!cell.!

While! lamin! A! plays! a! regulatory! role! and! there! are! !

different! mutations! of! the! genes! that! give! rise! to! different! So! from! a! chromosome! territory! you! can! have! the! looping!

diseases.! So! you! have,! first! of! all! the! muscle' diseases,! the! out! to! reach! some! transcription! or! repression,! so! the!

muscle! wasting,! dystrophy,! but! also! cardiomyopathy! and! lipodystrophy! that! is! alteration! of! chromatin!is!highly!dynamic.!

the!fat!tissue,!neuropathy!and!progeria!that!is!accelerated!aging.!! !

! 73! ! 74!

When! you! have! the! product,! you! have! interaction,! but! this! in! an! average! in! the! cell!

Techniques ! population;!if'you'don’t'see'the'product,'it'could'also'be'that'the'interaction'happens'but'

! it'very'rapid'and'dynamic.!Even!when!you!see!an!interaction,!you!cannot!say!that!100%!

FISH * of!the!cells!have!this!interaction.!That’s!why!then!you'need'to'validate'with'FISH!and!to!

This!technique!enables!to!visualize!at!the!same!time!two!different!genomic!loci,!even!if!they! consider!how!many!cells!in!a!population!present!that!interaction.!

are!located!on!two!different!chromosomes.!So!we!can!visualize!the!interactions!between!these! !

two!loci,!and!also!make!a!quantitative!measure!(not!qualitative).! !

The!immuneSFISH!couples!the!immunestaining!with!FISH!in!order!to!observe!the!position!of! 01.03.17!!

genes!among!them!and!also!respect!to!a!nuclear!protein,!against!which!the!Ab!is!directed.! Circular*Chromosome*Conformation*Capture*(4C)*

Limitations!of!FISH:! With!the!advent!of!the!sequencing,!everything!has!changed!because!even!the!3C!now!is!not!a!

! Probes! must! cover! at! least! 20Kb! to! visualize! unique! sequences,! so! it! is! not! really! new!technology.!

precise,!because!in!20Kb!there!could!be!also!in!another!regulatory!flanking!elements;!! When!you!want!to!see!the!interactions!within!one!locus!with!the!rest!of!the!genome,!you!can!

! Resolution!→!the!two!sequences!in!analysis!should!be!50S100Kb!far!from!each!other,! use,!instead!of!3C,!the!4C.!The!difference!is!that!by!3C!you!can!see!the!interaction!between!two!

otherwise!of!course!they!are!coSlocalizing! regions!that!you!should!know!(you!should!know!exactly!the!sequence!to!decide!the!restriction!

! The!number'of'interactions'is'limited!because!you!have!a!certain!number!of!colours!and! sites!and!the!primers),!while!with!the!4C!you!start!with!your!region!of!interest,!that!is!used!as!

you!cannot!go!up!of!6!colours!and!you!need!also!the!microscope.! a!bait,!and!then!you!can!catch!all!the!interactions!with!the!genome.!So,!it!is!an!unbiased!scan!of!

! the!genome.!

Chromosome*Conformation*Capture*(3C)* !

With! 3C! you! can! really! measure! the! frequency! of!

interactions!between!two!fragments.!!

If!you!have!two!fragments!(black!and!grey)!that!are!

very!far!from!each!other,!you!can!take!the!nuclei!and!

cross=link'them'with*paraformaldehyde.!! *

The! paraformaldehyde! interacts! with! the! amine! As! for! the! 3C,! you! have! a! crossSlink! that! can! freeze! the! interaction! between! different!

group! and! forms! a! crossSlink! between! all! these! fragments! that! are! far! from! each! other! in! the! genome.! Then,! the! principle! is! that,! since! you!

groups,!so!it!freezes!the!structure!of!chromatin,!both! have! exactly! the! same! restriction! sites,! upon! the! dilution! in! a! big! volume,! you! do! the! reS

at!DNA!level!and!protein!level,!creating!a!network!of! ligation,!so!favouring!these!circular!ligations.!Until!here,!it!is!exactly!as!the!3C.!!

proteinSDNA!interactions.!! But! then,! considering! that! we! know! the! black! sequence,! but! not! the! one! in! grey,! you! can!

If'the'two'fragments'were'close'to'each'other'in'the'nuclear'space,'for'sure'they'will'be'cross? design' the' primers' at' the' borders' of' your' sequence! that! go! toward! the! external! part! of! the!

linked'together.!! fragment.!Then,!you!amplify'the'unknown'product,!without!knowing!its!sequence.!!

Then!you!make!an!enzymatic* digestion.!If!you!want!to!study!specific!fragment!of!the!DNA,! Then!these!products!can!be!sequenced!or!can!be!cloned!in!plasmid!and!then!sequenced.!

first!of!all!you!have!to!study!which!enzyme!use,!because!it!should!cut!in!the!region!of!interest.!! The!efficiency!is!not!very!high.!

After!the!digestion,!you!dilute'a'lot!the!product!to!avoid!that!these!fragments!ligate!with!other! !

fragments!that!are!not!crossSlinked:!the!final!volume!is!10mL,!so!it!is!a!very!big!reaction!and! Chromosome*Conformation*Capture=on=Chip*(4C)*

you!need!a!lot!of!enzyme!so!is!an!expensive!experiment.! !In! that! case,! the! principle! is! exactly! the! same,! but! you! add'

Then!you!perform!a!ligation!forming!chimeric!products!(grey+black)!of!regions!that!are!not! one'step'that'is'another'digestion.'

contiguous!on!chromosomes:!the!10mL!volume!favours!the!ligation!only!between!fragments! You! have! your! bait' and' you' know' the' dimension,! but! you!

that!are!crossSlinked!together.! don’t! know! the! dimension! of! all! the! other! fragments!

Finally!using!specific!primers!close!to!the!restriction!site,!we!can!make!an!amplification!of!the! interacting!with!your!fragment!of!interest.!So,!you!can!have!

chimeric!products:!if!they!were!interacting!you!will!have!amplification,!otherwise!you!won’t! even!very!big!fragments:!with!this!type!of!technology,!if!you!

have!any!amplification.!! do! a! ligation! of! the! fragment! that! is! more! or! less! the! same!

! size! of! the! fragment! of! interest,! is! ok;! but! when! you! have! a!

Limitations!of!the!3C:! fragment! that! is! bigger! than! you! fragment! of! interest,! the!

! We!should!know'the'region!you!are!studying!to!choose!the!restriction!enzyme!and!the! plasmid!is!very!big!and!you!cannot!clone!it.!

primers! So,!you'are'favouring'the'detection'of'some'interactions'at'the'

! The!restriction'site'should'be'well'represented!in!those!regions! expense'of'other'interactions.!!

! It! requires! quite'a'lot'of'cells'(40! million! of! cells)! but! now! you! can! start! with! even! 5! With! this! technology,! upon! reSligation,! you! digest! again! the!

million!cells!if!you!want!to!see!a!limited!amount!of!interactions.!Because!if!you!want!to! fragment! with! an! enzyme! that! is! very! frequent.! So,! this!

do!a!scan,!you!need!a!quantity!of!material!to!amplify! enzyme!can!cut!really!a!lot!in!the!genome.!

! The! frequency'of'the'cross?linking'is'an'average'of'the'cross?linking.! So! you! can! see! an!

interaction,!but'you'cannot'see'the'non?interaction.!

! 75! ! 76!

First!of!all,!you!don’t!need!to!have!the!ligation!in!all!sites.!You!should!have!a!ligation!only!in! HiC *

one!site!so!you!can!still!digest!with!the!second!enzyme.! It!is!used!to!study'the'genome'conformation'without'specific'regions.!

And!second,!you!create!a!very!small!fragment,!your!region!of!interest!and!another!region!that!

is!variable!because!it!depends!where!the!second!enzyme!will!cut,!but!not!so!big.!

Then,!you!make!the!reSligation!and!then!the!principle!is!the!same.!

If!the!red!is!the!known!region!(the!bait),!you!design!the!primers!and!you!amplify!this!product.!

'

4C'applications'in'the'medical'field'

This!4C!was!then!applied!also!in!the!medical!field!because!it!can!detect!some!translocations.!

For!example,!in!the!control!you!have! !

your!bait!and!this!type!of!interaction.!! You!have!to!crossSlink!all!the!sequence,!cut!with!restriction!enzyme,!fill!it!with!the!biotin,!reS

When! you! have! a! translocation,! you! ligate! and! immunoprecipitate! with! the! biotin! (without! protein! and! without! aiming! to! a!

really! break! this! type! of! peak! and! specific!sequence);!then!you!amplify!and!sequence.!!

finally!you!will!find!a!half!of!the!pick! Of!course,!some!parts!of!the!genome!won’t!be!cut!as!the!digestion!efficiency!is!not!100%!even!

on! the! other! chromosome.! The! shape! of! the! pick! is! very! important! because! you! will! have! a! because!you!have!crossSlink,!so!all!the!proteins!can!mask!some!restriction!sites.!!

probability!to!have!a!ligation!with!the!flanking!sequence!that!is!lower!and!then!go!up!and!then! Finally,!you!obtain!this!result.!

lower.!Of!course,!the!shape!depends!also!on!the!length!of!the!fragment.!! In!red!you!have!the!interactions.!!

When!you!detect!the!translocation,!you!will!find!half!of!the!pick!in!one!chromosome!and!the! If! you! measure! one! chromosome! on! itself,! you!

other!half!on!the!other!chromosome.!So,!you!know!that!you!have!a!translocation!and!where!is! have!the!diagram!that!is!completely!red.!This!is!a!

the!translocation.! specular' graph' so' you' can' consider' only' a' half' of'

This!can!be!useful!especially!in!cancer!where!you!have!a!lot!of!events!of!translocation.!! this'plot.'

! If! you! don’t! have! interaction,! you! obtain!

Limitations!of!4C!technology:! something!like!in!the!second!graph.!!

! You!should!have'a'bait,!so!you!should!have'a'region'that'you'are'interested!to!study!and! !

you!should'know'the'sequence'of'this'region! !

! The!restriction'sites'should'be'well'represented'in'this'region!! They! found! that! there! are! some! subSchromosomal!

! You!need'a'lot'of'cells,'more'than'in'3C! compartments,! that! they! called! topologically!

! You! don’t' know' what' happen' cell' by' cell,! so! in! each! nucleus! which! interactions! take! associated!domains!(TADs).!

place! This!is!important!because!it!is!the!first!proof!that!the!

* genome! is! organized! in! an! ordered! structure,! not!

* random.!

ChIP=loop* Interactions!by!HiC!must'be'confirmed'FISH'or'3C.'

This!technology!is!useful!when!you!want!to!see! interactions'among'fragments'mediated'by'a' !

protein.' !

You!immunoprecipitate'the'protein!and!then!you!make!this!type!of!ligation:!so!you!catch'only' They! found! that! the! genome! is! not! folded! randomly,! but! form! these!

the'interactions'that'depend'on'this'protein.! specific! structures! where! all! the! sequences! that! have! more! or! less! the!

In! this! paper,! they! did! it! with! polycomb! complexes.! They! crossSlinked,! immunoprecipitated,! same!degree!of!transcription!are!clustered!together.!

ligated!and!then!they!identified!their!sequence.! !

! !

! Limitations!of!HiC!

Limitations!of!ChIPSloop:! ! The'restriction'sites'should'be'well'represented'

! Restriction'sites'should'be'well'represented' ! You'need'a'lot'of'cells'

! You'need'approximately'40'million'cells' ! You'don’t'know'what'happen'cell'by'cell.!

! You' don’t' know' the' variability' in' the' !

population.' *

! *

! *

! *

! *

! *

! 77! ! 78! 24.02.2017!

ChIA=PET* * Dynamics*of*DNA*repetitive*elements*in*cell*programming,*differentiation*

(Chromatin'Interaction'Analysis'by'Paired?End'Tag'Sequencing)'

It! is! very! similar! to! the! ChIPSloop! assay:! you! immunoprecipitate! the! protein! and! then! you! and*disease*

make! the! ligation;! while! in! the! previous! one,! you! first! make! the! ligation! and! then! you! What!is!considered!junk!DNA,!is!actually!a!portion!of!the!genome!with!epigenetic!functions,!

immunoprecipitate!your!protein.! that!regulates!the!transcriptome.!

It!uses!some!adapters,!instead!of!biotin.!! !

Finally,!you!have!again!the!chimerical!products!and!your!sequence.! The!human!genome!is!composed!by!23!couples!of!chromosomes.!

Thinking!about!the!evolution,!it!was!thought!that!human!genome!was!the!biggest,!but!it's!not!

the!true;!this!was!the!first!observation!for!the!starting!of!the!characterization!of!the!genome.!

The! second! observation! is! about! the! complexity! of! the! genome! that! does! not! reflect! the!

composition!of!the!protein!coding!genes:!in!the!human!genome,!the!non?coding'portion'is'the'

biggest'one.'

!

Data! must! always! be! validated! with! FISH! analysis:! FISH' is' complementary' to' these' C' In! bacteria! there! are! linear! correlations! between! composition! of! protein! coding! genes! and!

technologies'because'it'gives'you'also'the'variability'in'a'cell'population.'' dimension!of!the!genome,!but!this!is!not!true!for!the!rest!of!the!species.!

' !

With! the! ChIASPET,! it! was! found! that! this! estrogen! receptor! If!you!look!which!is!the!big!portion!of!the!genome,!you!see!that!there!are!repetitive'sequences.!

forms!this!specific!conformation!when!activated.! You!should!have!technologies!able!to!define!the!single!units!and!it's!complicated!because!of!

! the!homology!of!sequence!(more!than!99%)!and!it's!very!difficult!to!have!a!real!idea!of!how!

Limitations!of!ChIASPET! many!they!are.!

! You! need! 100' million' cells! (you! can! do! it! only! on! cell! Sequences!can!be!divided!on!the!base!of!their!repetitions:!!

culture,!not!in!tissue)! ! Non'repetitive'sequences'

! You!don’t'know'about'the'variability.! ! Moderate'repetitive'sequences'

* ! High'repetitive'sequences'

** '

* E.Coli!doesn't!have!any!of!these!repetitive!elements,!so!100%!of!the!genome!is!composed!by!

! unique!sequences.'

! Mouse!is!more!similar!to!the!human!and!there!is!a!good!portion!of!repetitive!elements.!

! !

! If!you!want!to!categorize!the!human!genome!on!the!base!of!these!observations!(so,!if!they!are!

! repetitive! elements! or! not)! you! have! to! consider! the! division! in! gene! sequences! and! extra!

! gene!sequences:!

! ! Gene!sequences!→!25%!of!the!genome,!of!which!coding*sequences!are!1.5%!

! ! NonScoding!sequences!→!introns,!gene!fragments,!pseudogenes,!non!coding!RNAs.!

! !

! Extra?gene'sequences.'

! They!are!divided!into:!

! ! Clustered'(tandem)'repeats'

! ! Interspersed'repeats'

! 67=70%!of!the!human!genome!is!composed!by!repetitive*elements.!

! !

! Tandem*repeats*

! The! tandem' array! is! that! portion! of! the! genome! that! is! highly' repetitive,! meaning! that! you!

! have!hundreds!of!thousands!of!copies!that!compose!an!array.!You!don't!know!really!say!the!

! exact!composition!of!the!array!in!the!genome.!

! The!tandem!arrays!are!divided!in!two!categories:!!

! ! Satellites'→! structural'role'in! the! chromosome! composition;! the! most! frequent! in! the!

! genome!is!the!alpha!satellite.!They!are!divided!for!the!length!of!the!repetition.'

! ! Variable'number'tandem'repeats.'

! !

! !!

! 79! ! 80!

Satellite*DNA!! Another! disorder! is! associated! with! a! nonScoding! potential! of! these! kind! of! repeats! is! the!

It!is!the!main!component!of!functional!centromere!with!some!diversity!in!the!classes.!! fragile'X'syndrome.!

!

For!example:!the!α!satellite!composes!the!centromere!fold!the!chromosomes,!while!β!satellite! Interspersed*repeats*–*Transposable*elements*

is!present!in!some!chromosomes,!it!is!peculiar!of!acrocentric!chromosomes.! These!copies!are!generated!by!the!copy!and!paste!mechanism.!The!most!common!uses!RNA!

Then,! there! are! others! that! are! present! in! most! chromosomes! with! big! differences! in! the! intermediate!to!be!copied!and!they!are!retrotransposomes.!!

length!array.!Each'individual'is'polymorphic'for'these'kind'of'satellites.' Transposable!elements!can!be!divided!in:!

! Unable'to'encode'for'the'retranscriptase! retrotranscription! of! RNA! intermediate! to!

In!the!acrocentric!chromosomes!β!satellites!surround!the!ribosomal!DNA,!which!is!a!stretch!of! →!

• create!new!copies!of!DNA,!then,!pasted!in!the!genome;!

tandem!genes,!that!are!repeated!in!50S70!copies.!This!region!is!actively!transcribed.! Able'to'encode'for'the'retrotranscriptase.!

In!the!human!genome!rDNA!is!present!in!10!chromosomes!(5!pairs):!the!big!acrocentrics,!that! •

This!means!that!they!can!have!evolutionary!origin!viral!or!not!viral.!

are!13S14S15!and!the!little!acrocentrics!21S22.! !

! SINE*(Short'interspersed'nuclear'elements)'

! They!are!new!evolved!retrotransposomes,!that!were!not!present!during!evolution.!

Variable*number*tandem*repeats* In!mice!there!are!just!some!SINE!while!human!genome!and!primate!genome!are!very!rich!of!

They!are!divided!on!the!bases!of!dimension!of!the!single!units!that!compose!the!satellites,!but! SINE!elements.!

repetitions!are!not!so!big;!they!are!polymorphic.!! ALU'is'human'specific:!it!is!the!most!frequent!in!the!genome.!

They! are! well! characterized! in! the! human! genome! and! there! are! many! techniques! that! can! SINE! elements! are! not! autonomous! (not! protein! coding! potential)! and! they! need! a! LINE!

give!you!an!idea!of!the!dimension.!! machinery!to!retrotranspose!themselves.!

They!are!inherited'and'they'can'be'a'marker'of'genetic'profile.! !

On!the!bases!of!the!repetitions!they!are!divided!in:!! !

Macrosatellites!

• LTR!

Minisatellites!→!in!general,!GC!rich;!they!can!vary!from!6S100!bp,!so!it's!very!simple!

• LTR!have!a!viral'origin,!indeed!they!are!speciesSspecific.!!

to!be!detected,!because!they!are!little!enough!to!be!amplified.! LTR!transposomes!have!a!structure!that!remembers!that!of!the!virus,!but!they!accumulated!so!

More!than!1000!locations!in!the!human!genome!and!they!have!preferential!location!is! many!mutations!that!are!not'able'to'propagate'themselves'like'virus'particles.!

the!telomeric!–!sub!telomeric.! !

They!are!preferentially!located!in!centromere,!subtelomere!regions.! LINE*(Long'Interspersed'Nuclear'Elements)'

The! most! famous! minisatellite! is! the! sequence! of! the! telomeres,! that! is! also! They!are!the!only!class!of!repeated!transposable!elements!autonomous!in!the!human!genome.!

evolutionary!conserved.! In!the!structure!is!present!ORF2!that!encodes!for!the!retrotranscriptase!and!has!a!promoter!

Microsatellites!→!short!tandem!repeats!or!simple!sequence!repeats.!

• that!produces!a!mRNA.!

They!are!studied!for!two!reasons:!! The!other!retrotransposomes!use!the!ORF2!of!the!LINE!to!retrotranspose!themselves.!

1. Forensics!→!the!combination!of!STR!is!a!way!to!identify!a!person.!Just!with! !

few!molecules!of!DNA,!you!can!amplify!with!PCR,!for!example!a!combination! LINE!are!divided!in:!!

of! 16! STR! stranded! along! the! chromosomes.! The! perfect! combination! of! ! Full! length! elements! some! thousands.! Some! of! them! are! evolutionary! young!

polymorphic!STR!identified!a!person,!but!with!a!percentage!of!identification! →!

elements!and!are!active!

(but!it's!so!high!that!is!considered,!basically,!the!true!identity).!It's!used!for! ! Truncated!elements!!

paternity! test,! because! you! can! reconstruct! the! maternal! and! paternal! !

chromosomal!origin!of!the!child.! Usually!the!promoter!of!LINE!is!methylated,!but!when!it's!unmethylated!and!a!transcription!

2. Association'with'diseases'→! some! known! diseases! are! caused! by! mutations! factor!binds!it,!the!transcriptional!machinery!can!recognise!this!promoter!and!transcribe!the!

in! STR.' They! are! divided! in! two! classes! based! on! the! localization! of! the! mRNA.!!

mutation:! when! the! repetition! of! three! nucleotides! is! coding! or! it's! not! !

coding.' LINE1! is! the! most! common! and! active! class! of! LINE! elements! in! the! human! genome.! These!

! elements!are!full'of'regulatory'sequences.!

! Indeed!there!are!mRNAs!that!have!splicing!recognition!sequences!that!can!include!repeated!

Huntinghton!disease!is!caused!by!coding!repetitions!that!encode!for!polyglutamine!stretches;! structures!because!they!can!participate'in'the'alternative'splicing'machinery'and'can'create'a'

when!this!repetition!are!higher!than!a!threshold,!the!expression!of!the!protein!becomes!toxic! variability'in'the'mRNA.!

because!it!accumulates!in!the!cells.! They!can!also!produce!ncRNAs.!

! !

A!nonScoding!disease!is!myotonic'dystrophy,!when!you!have!a!CAG!repetition!in!the!5'!UTR!of!

the!mRNA!and!in!this!case!the!repetition!itself!causes!a!secondary!structure!in!the!mRNA.!

! 81! ! 82!


PAGINE

42

PESO

20.75 MB

AUTORE

_ariiel

PUBBLICATO

7 mesi fa


DESCRIZIONE APPUNTO

Programma
Non-coding regulatory rnas
- Small non-coding RNAs (snRNA, snoRNA, siRNA, microRNA, piRNA): definition, classification, structure, mechanism of action and physio-pathological examples.
- Long non-coding RNAs: definition, classification, structure, mechanism of action and physio-pathological examples.

Dynamics of the repetitive elements of dna in cell identity, differentiation, pathologies and their epigenetic role.
- Tandem repeats, satellite DNA, VNTR, Retrotrasposons, SINEs, LINEs: definition, classification, structure, mechanism of action and physio-pathological examples.

3D genome architecture
- Genome hierarchy and organization.
- Models for the genome tridimensional organization.
- Functional role of nuclear domains and chromosome territories.
- Techniques for the study of 3D genome structure.

Next generation sequencing: applications and data analysis
- NGS: Principles and technologies.
- NGS applications: whole genome sequencing; transcriptomics; capture sequencing (exome sequencing and custom target); epigenetics.
- Single cell transcriptomics.
- Elements of bioinformatic for NGS data analysis.

Genome engineering
- Gene trapping, gene targeting, conditional knockout.
- Homologous recombination, artificial restriction enzymes: Zinc Fingers Nucleases, TALEN, CRISPR/Cas9.


DETTAGLI
Corso di laurea: Corso di laurea magistrale in biotecnologie mediche e medicina molecolare
SSD:
Università: Milano - Unimi
A.A.: 2017-2018

I contenuti di questa pagina costituiscono rielaborazioni personali del Publisher _ariiel di informazioni apprese con la frequenza delle lezioni di Molecular biology applied to biotechnology e studio autonomo di eventuali libri di riferimento in preparazione dell'esame finale o della tesi. Non devono intendersi come materiale ufficiale dell'università Milano - Unimi o del prof Pagani Massimiliano.

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