CRISPR-Cas9

CRISPR - Cas9
 CRISPR – Clustered Regularly Interspaced Palindromic Repeats
 CRISPR is a family of DNA found in genomes of prokaryotic organisms like bacteria and archaea.
 CRISPR was first found in E. coli genome in 1987 by Ishino in Osaka University.
 CRISPR - Cas9 genome editing method was developed by Emmanuelle Charpentier and Jennifer Doudna
in 2012 for which they were awarded with Nobel Prize in 2020.
 CRISPR together with Cas genes form an adaptive immunity which provides resistance against viral
infections.
 CRISPR-Cas system is a highly adaptive and heritable resistance mechanism that incorporates short
sequences from viruses and other mobile genetic elements into the host’s CRISPR locus to be transcribed
and processed into small RNAs that guide the destruction of invading nucleic acids.
Functioning of Type II CRISPR-Cas System in Bacteria
 Type II CRISPR - Cas system involves single multi-functional Cas 9 protein.
 In the endogenous CRISPR-Cas9 system three components are necessary for target cutting:
a. Cas9 protein,
b. CRISPR RNA (crRNA) and
c. transactivating crRNA (tracrRNA),

 The type II CRISPR - Cas system comprises three stages:

I. Acquisition of CRISPRs
II. crRNA biogenesis
III. Interference with invading DNA
I. ACQUISITION OF CRISPRs

Phage DNA

Cas Nuclease

Protospacer sequences

Incorporated into CRISPR Locus

As a new spacer

PAM – Protospacer adjacent motif

PAM Sequence depends on the species of Cas9
proteins.
Ex-
Streptococcus pyogenes - 5’-NGG-3’
Streptococcus thermophilus – 5’ NGGNG – 3’
Hryhorowicz et.al (2017)
II. BIOGENESIS OF crRNA

CRISPR locus
Transcription

Pre- crRNA

tracrRNA hybridises to repeat

sequences of pre-crRNA

RNAase III

mature crRNA

Hryhorowicz et.al (2017)

III. INTERFERENCE WITH INVADING
DNA
 mature crRNA guides Cas9 protein to
the complementary foreign nucleic
acids, triggering degradation of the
DNA sequences of invading phages.

 The Cas9 protein contains HNH and

RuvC nuclease domains, which cleave
the DNA complementary strand and
the non-complementary strand,
respectively, introducing double strand
break repairs.

Hryhorowicz et.al (2017)

EXPLOITING CRISPR-Cas9 FOR GENOME ENGINEERING

 The guide sequence within CRISPR spacers corresponds to foreign viral genomes constituting the form of the
acquired immunity of bacteria, but can easily be substituted by a sequence of interest to target the Cas9
protein.

 Cas9-generated site-specific DNA double-strand breaks induce endogenous cellular DNA repair processes,
which can be exploited to engineer the genome.

 Double Strand Breaks are generally repaired by one of two pathways:

 Homologous directed repair (HDR) if the homologous template is available
 Nonhomologous end joining (NHEJ)
Engineered nuclease-induced genome editing. A double-stranded break (DSB) in the targeted sequence can be
repaired through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways.

Hryhorowicz et.al (2017)

APPLICATIONS

 CRISPR-Cas9 in the generation of animal models: CRISPR-Cas9 technology can be used for rapidly
generating targeted genome modifications in the germ lines of various model organisms, which will
significantly advance the functional genomics.
 CRISPR-Cas9 in correction of genetic disorders: For Example, correction in dominant Crygc gene mutation
in a cataracts mouse
 CRISPR-Cas9 in the treatment of infectious diseases : This system can be used for treating infectious
diseases by eradicating pathogen genomes from infected individuals. For example, studies have shown that the
CRISPR-Cas9 system can eliminate the HIV-1 genome.
 Non-nuclease Uses of Cas9 Protein: The CRISPR-Cas9 system can also be used to regulate the expression of
endogenous genes. Catalytically dead Cas9 (dCas9) protein with inactive RuvC and HNH nuclease domains
retains the ability to bind to target DNA and causes repression of the target gene by a steric block that stops
transcript elongation by RNA polymerase.
CONCLUSION
The CRISPR-Cas9 technology, an efficient, inexpensive, fast-to-design, and easy-to-use genomic editing tool,
has been rapidly applied in many fields, ranging from basic biology to translational medicine.
Cas9 mediated genome editing provides new therapeutic strategies for infectious diseases, wound healing and
tissue regeneration.
CRISPR - Cas9 system provides a wide approach to research and study genes and its functions.
REFERENCES
 Hryhorowicz, M., Lipiński, D., Zeyland, J. et al. CRISPR/Cas9 Immune System as a Tool for
Genome Engineering. Arch. Immunol. Ther. Exp. 65, 233–240 (2017).
https://doi.org/10.1007/s00005-016-0427-5

 Yang, X. (2015). Applications of CRISPR-Cas9 mediated genome engineering. Military Medical

Research, 2(1), 1-6.

 Redman, M., King, A., Watson, C., & King, D. (2016). What is CRISPR/Cas9?. Archives of Disease
in Childhood-Education and Practice, 101(4), 213-215.

 https://www.drugtargetreview.com/news/74139/nobel-prize-in-chemistry-awarded-to-scientists-who
-discovered-crispr-cas9/
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