8/9/2019 CRISPR.pptx

1/8

CRISPR/Cas9

8/9/2019 CRISPR.pptx

2/8

 CDK9, CDK12 and CDK13 knockout

CRISPRConsist of several non-contiguous direct repeats sa!e se"uence# se$arated %&spacers  of varia%le se"uencesco!$le!entar& to seg!ents of

ca$tured viral and $las!idse"uences#'

(( CRISPR clustered regularl& inters$aced s)ort $alindro!ic re$eats#

 *&$e I *&$e II *&$e III

8/9/2019 CRISPR.pptx

3/8

fter infection of t)e %acteria

8/9/2019 CRISPR.pptx

4/8

((*)e P+ $rotos$acer ad.acent !otifs# se"uence is re"uiredto i!!ediatel& follo t)e target D0+ locus, %ut t)at its not a

$art of t)e 2-nt guide se"uence it)in t)e sgR0+'

eno!e engineering

8/9/2019 CRISPR.pptx

5/8

 *&$e II s&ste!s rel& on a single $rotein, Cas9, for t)e cleavageste$' Cas9 re"uires %ot) t)e crR0+ and t)e tracrR0+ to functionor c)i!eric sgR0+# and cleaves D0+ using its do!ains

-505 nuclease do!ain cleaves t)e co!$le!entar& strand'

- RuvC-like do!ain cleaves t)e non-co!$le!entar& strand'

6ot) do!ains $roduce dou%le-strand %reaks, and se$aratel& t)e&can $roduce single-strand %reaks nickase#

8/9/2019 CRISPR.pptx

6/8

D0+ da!age re$air

0on)o!ologous end-.oining NHEJ# !ec)anis!, )ic)is error $rone and introduces fra!es)ift indels,resulting in a knockout inactivation of t)e gene'

5ig)-7delit& 5DR )o!olog&-directed re$air#, )ic) can

generate $recise, de7ned !odi7cations at a targetlocus in t)e $resence of an e8ogenousl& introducedre$air te!$late'

1

2

8/9/2019 CRISPR.pptx

7/8

CDK knockout

RISPR nickase - co!$onents2 gR0+Cas9 endonucleaseD0+ construct it) selecta%le !arker#

Reduces o: target e:ects

8/9/2019 CRISPR.pptx

8/8

s

sign t)e gR0+ using a softare#"uence t)e cells D0+$are t)e gR0+ vectorsnsfect t)e cells it) t)e gR0+, t)e Cas9 vector and t)e D0+ conn7r! engineered cells