A short presentation to introduce CRISPR/ CAS with students

2. WHAT IS CRISPR-CAS?
• CRISPR (Clustered Regularly Interspersed Short palindromic Repeats)
associated protein-9 nuclease (Cas 9) from S. pyogenes –a new tool
• Initially identified as a part of adaptive immunity in bacteria and archaea
against invading genetic material
• Demonstrated in S. thermophilus to acquire immunity against bacteriophage by
integrating a part of its genome into the CRISPR locus

3. MECHANISM
Invading DNA from viruses or plasmids is cut into small fragments and
incorporated into CRISPR locus in between a series of short repeats
(around 20bps)
The loci are transcribed and transcripts are processed to generate small
RNAs (crRNA) used to guide effector endonucleases that target invading
DNA based on sequence complementarity

4. CAS9
• Out of three CRISPR mechanisms type II is most efficient requiring only one
Cas protein Cas9 for gene silencing
• Cas9 participates in processing of crRNA and is responsible for destruction of
target DNA
• The function of Cas9 relies on two nuclease domains- RuvC like nuclease
domain at the amino terminus and HNH like nuclease domain in the mid region
of the protein
• During target DNA destruction the RuvC and HNH domains cut both DNA
strands generating DSBs at sites defined by 20nt sequence within an associated
crRNA transcript.
• The HNH domain cleaves the complementary strand while RuvC cleaves the
non complementary strand.

5. TRANS ACTIVATING RNA
• Site specific DNA recognition and cleavage requires that Cas9 is complexed with
crRNa and a separate trans acting RNA that is partially complementary to the
crRNA
• The tracrRNa is required for crRNA maturation from a primary transcript
encoding multiple pre-crRNAs in the presence of Cas9 and RNAseIII

6. PROTOSPACER ASSOCIATED MOTIF
• Endonuclease activity of Cas9 also requires that a short conserved sequence of
2-5 nt is present immediately 3’ to the crRNA complementary sequence (PAMs)
• In the absence of PAMs even fully complementary sequence is ignored by Cas9

7. USED AS A TOOL
A simplified two component system has been developed by combining crRNA and
tracrRNA into a single guide RNA which has been shown to be effective with
Cas9
Three different variants of Cas9 nuclease are used in genome editing protocols

8. VARIANTS OF CAS9
• Wild type Cas9 which site specifically cleaves double stranded DNA resulting in
activation of DSB repair machinery
• DSBs can be repaired by Non-Homologous End Joining machinery resulting in
indels-insertions or deletions
• It can also be repaired by homologous repair pathway
• Cas9D10A mutant has only the nickase activity so it can cleave only one strand
of DNA and when provided with homologous repair template can undergo HDR
pathway
• dCas9 variant is nuclease deficient. Mutations H840A in HNH domain and
D10A in RuvC domain inactivate cleavage activity. This variant is used to
sequence specifically target any region of the genome and can be used for
visualization or modification

9. REFERENCES
PrimRose
www.google.com
NEB site