CRISPR/Cas9

• It is an RNA-guided endonuclease that specifically targets

DNA sequences via nucleotide base pairing

• CRISPR stands for Clustered Regularly Interspaced Short

Palindromic Repeat DNA sequences

• The CRISPR/Cas system was first described as an

adaptive immune system in bacteria and archaeons,
Type II CRISPR systems consists of
crRNAs

tracrRNA

Cas9 protein
 CRISPR-RNA (crRNA)

 A large RNA is transcribed from the CRISPR

repeat/spacer array(CRISPR locus ), the so called pre-
CRISPR-RNA

 It contains multiple spacers inserted between repeats

 It is cleaved into mature crRNAs that contains the

spacer flanked by a portion of an adjacent repeat

What is the spacer

DNA segments from foreign genetic elements (such as viruses

and plasmids) are integrated into a CRISPR array
Trans-activating CRISPR-RNA (tracrRNA)

• a small, non-coding RNA which is

encoded in the vicinity of the
CRISPR array and cas genes
• The tracrRNA contains an antirepeat, i.e. a
region that is(partially) complementary to the
repeat in a pre-crRNA

It has a role in crRNA

trimming into a mature

Spacer acquisition
• The programmable
crRNA and fixed
tracrRNA are fused to
form an sgRNA (single-
guide RNA), which
directs Cas9 to the
desired site and
catalyzes the cleavage
of both DNA strands
effectively
CRISPR-associated nuclease 9(Cas9)

• It contains the RuvC and HNH(His-Asn-His) nuclease domains

• Cas9 break the double-stranded DNA site primarily located 3 bp
upstream of (PAM) sequences in the target DNA .
• The HNH domain cuts a complementary strand of crRNA, whereas
• the RuvC-like domain cuts an opposite strand of dsDNA
 Type II CRISPR systems
Trans-activating
CRISPR-associated
CRISPR-RNA (crRNA) CRISPR-RNA
nuclease 9(Cas9)
(tracrRNA)
A large RNA is transcribed It contains the RuvC and HNH(His-
from the CRISPR Asn-His) nuclease domains
It is transcribed from the
repeat/spacer array(CRISPR
locus ), the so called pre- tracrRNA locus.
CRISPR-RNA (pre-crRNA) Cas9 break the double-stranded DNA
site primarily located 3 bp upstream of
(PAM) sequences in the target DNA .
Thereafter, pre-crRNA
It is complementary to the The HNH domain cuts a complementary
and pre-tracrRNA are
repeat domains of the strand of crRNA, whereas
trimmed to their final
pre-crRNA and binds to it
length by the RNaseIII the RuvC-like domain cuts an opposite
via base pairing
enzyme strand of dsDNA

Interaction between crRNA and tracrRNA directs Cas9 to

recognize the specific DNA sequence complementary with
the protospacer
The PAM(the protospacer adjacent
motif)
• It is a short specific sequence following the target DNA
sequence that is essential for cleavage by Cas nuclease.
• The acquired spacer sequences are highly similar to each other at regions
of (PAMs)
• The PAM is about 2-6 nucleotides downstream of the DNA
sequence targeted by the guide RNA and the Cas cuts 3-4
nucleotides upstream of it.
• In S. pyogenes, for example, Cas9 recognizes a 5′-NGG-3′ PAM
(where “N” can be any nucleotide base). However, the spacers
in its CRISPR array are coded by 5’-GTT-3’, so the Cas9 cannot
cut the bacteria’s own genome.
Development history of the CRISPR/Cas9-based gene editing tools
 depending mainly on
the organization of the

two classes of CRISPR systems nuclease portion

Class 1.
• contains type I, IV and type III CRISPR systems that
are commonly found in Archaea

Class 2
• contains type II, V, and VI CRISPR systems.
• the most widely used one is the type II CRISPR-
Cas9 system from Streptococcus pyogenes.
Because of the simple NGG PAM sequence
requirements,
 Process of CRISPR-Cas acquired immune
system
• The invading DNA is recognized by Cas
.. Ad proteins, fragmented and incorporated
ap into the spacer region of CRISPR, and
tat stored in the genome.
ioE
nx
p • Pre-crRNA is generated by
r transcription of the CRISPR region and
e is processed into smaller units of RNA,
sI named crRNA.
sn
it
oe • . By taking advantage of the homology of the
nr spacer sequence present in crRNA, foreign
f DNA is captured, and a complex with Cas
protein having nuclease activity cleaves DNA.
e
r
e
n
c
e
 Genome editing
• It is a technique which introduces desired changes into genomes DNA
by

Insertions Deletions(indels) Base substitutions

Genome editing comprises various
techniques such as

The zinc finger nucleases (ZFNs)

Transcription activator-like effector (TALE) protein

CRISPR/Cas9
CRISPR/Cas as an universal gene editing system

knock-ins
gene (KIs).
knock-out the complex binds via sgRNA
base pairing to the target double strand breaks
sequence. Cas9 will make a made by Cas9 are
double strand break into the known to stimulate
target gene and the often faulty homologous
repair causes small insertions recombination.
or deletions (indels).

By presenting specific
This so called non-homologous repair templates, it is
end joining (NHEJ) is used to also possible to
create knock-outs of genes introduce specific
(KOs) modifications into the
target
Examples for other applications of CRISPR/Cas

• Inactivation of either one of the two nuclease domains of Cas9 leads to

a single-strand breaking Cas9 (nickase, nCas9). Tandem use of two
parallel nCas9 proteins was shown to improve targeting specificity
• If both nuclease domains are inactivated, a Cas protein results which is
unable to cleave DNA but still binds to the specific target sites (dCas,
Fig. 5). Such dCas variants can be fused for example with activators or
repressors of gene expression or with epigenetic markers or
fluorescent proteins. The latter may be used as novel diagnostic tools
to detect viruses or infectious bacteria in the future.
Base editors for “second generation
CRISPR/Cas genome editing
• The successful fusion of cytidine and adenine desaminases to dCas9
turned an epigenetic marker again into a genetic marker which works
without cutting DNA.
• These enzymes induce specific nearby base conversions (C→T;
A→G) and stop codons were introduced successfully by this technique
into target genes. Base editors may pave the way to site-directed
mutagenesis of the genome, the so called second generation
CRISPR/Cas genome editing.
Problems and drawbacks of CRISPR/Cas
• The most obvious drawbacks of CRISPR/Cas are off-target effects resulting from the fact
the target-specific moiety in the sgRNA is only 20 bases long.
• One of the technical challenges is the delivery of such tools into living cells and
organisms. The large size of current Cas proteins creates a major challenge in their
packaging into AAV vectors.