CRISPR/Cas9

CDK9, CDK12 and CDK13 knockout

CRISPR

Type I
Type II
Type III

Consist
of
several
noncontiguous
direct
repeats
(same sequence) separated by
spacers of variable sequences
(complementary to segments of
captured viral and plasmid
sequences).

** CRISPR (clustered regularly interspaced short palindromic repeats)

After infection of the bacteria:

Genome engineering

**The PAM (protospacer adjacent motifs) sequence is required

to immediately follow the target DNA locus, but that its not a
part of the 20-nt guide sequence within the sgRNA.

Type II systems rely on a single protein, Cas9, for the cleavage

step.Cas9 requires both the crRNA and the tracrRNA to function
(or chimeric sgRNA) and cleaves DNA using its domains:
- HNH nuclease domain: cleaves the complementary strand.
- RuvC-like domain: cleaves the non-complementary strand.

Both domains produce double-strand breaks, and separately they

can produce single-strand breaks (nickase)

DNA damage repair:

1 Nonhomologous end-joining (NHEJ) mechanism, which

is error prone and introduces frameshift indels,

resulting in a knockout inactivation of the gene.
2 High-fidelity HDR (homology-directed repair), which can

generate precise, defined modifications at a target

locus in the presence of an exogenously introduced
repair template.

CDK knockout

CRISPR nickase - 4 components:

- 2 gRNA
- Cas9 endonuclease
- DNA construct (with selectable marker)

Reduces off target effects

ps

esign the gRNA (using a software)

quence the cells DNA
epare the gRNA vectors
ansfect the cells with the gRNA, the Cas9 vector and the DNA cons
onfirm engineered cells