CRISPR-Cas9 is a revolutionary genome editing tool that allows targeted modifications to DNA. It utilizes the Cas9 endonuclease enzyme, which is guided to a specific location in the genome by a short RNA molecule. When the Cas9 enzyme cuts the DNA, it triggers the cell's repair mechanisms which can introduce changes to the genome at that location. CRISPR-Cas9 has significant advantages over previous genome editing techniques in terms of efficiency and ease of use. It holds promise for curing genetic diseases, advancing biomedical research, and improving crops and livestock. Future directions include optimizing delivery methods and enhancing the precision and control of genome alterations.

1. Development And Application of
CRISPR Cas9 for Genome Engineering
AJAY PRAKASH UNIYAL
M.Sc. Plant Science
Central University Of Punjab

3. INTRODUCTION
 Genome engineering refers to the process of making targeted modifications
to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g.
transcripts)
 Genome engineering with programmable nucleases depends on cellular
responses to a targeted doublestrand break (DSB).
 DNA double-strand breaks (DSBs) are potentially lethal to cells, which have
two broad classes of mechanisms to repair them: non-homologous end
joining (NHEJ) and homology-directed repair (HDR).

4. Figure: Hsu, Lander, Zhang: Development and Applications of CRISPR-Cas9 for Genome Engineering; Cell 157,
June 5, 2014

5. 1. Meganucleases
2. Zinc Finger Nucleases
3. Transcription Activator like Effector Nucleases (TALENS)
4. CRISPR / Cas 9 systems.
CRISPR/Cas9 unprecedented efficiency and stunning ease of use is what
makes it better.
Programmable Nucleases For Genome Engineering

6. CRISPR: Clustered Regularly Interspaced Short Palindromic
Repeats
Cas proteins: CRISPR-Associated Proteins
CRISPR systems are adaptable immune mechanisms used by many bacteria to
protect themselves from foreign nucleic acids, such as viruses or plasmids
(Sternberg et al., 2012)
It uses antisense RNA as the memory signatures for the past invasions
(Makarova et al, 2012)
What is CRISPR/ Cas9 ?

7. CRISPR loci typically consist of a clustered set of CRISPR-associated (Cas) genes
and the signature CRISPR array—a series of repeat sequences (direct repeats)
interspaced by variable sequences (spacers) corresponding to sequences within
foreign genetic elements (protospacers)
Source :C L Sun et al., 2013 doi: 10.1111/j.1462-2920.2012.02879.
CRISPR LOCI

8. (1) acquisition of foreign
DNA
(2) synthesis and maturation
of CRISPR RNA (crRNA)
followed by formation of
RNA-Cas nuclease protein
complexes
(3) target recognition by
crRNA and destruction of
foreign DNA by
Cas nuclease cleavage
http://dx.doi.org/10.1016/j.cell.2014.05.010
CRISPR/Cas the immune system of bacteria

9. Different CRISPR-Cas System in Bacterial Immunity
Class 1
Class 2
Type I : CRISPR Cas3
Type III : CRISPR Cas 10
Type II : CRISPR Cas9
The type I and type III systems use a large complex of Cas proteins for crRNA-guided
targeting (Andersson and Banfield, 2008)
However, the type II system requires only a single protein for RNA-guided DNA
recognition and cleavage (M. Jinek et al.,2012) —a property that proved to be
extremely useful for genome engineering applications

10. http://dx.doi.org/10.1016/j.cell.2014.05.010

11. Organisation and Domain Structure Of Cas 9 protein
 Bioinformatic analyses first identified Cas9 as a large multifunctional protein with
two putative nuclease domains, HNH (Tang et. al.,2012) and RuvC-like domain.
 Cas9 uses its HNH domain to cleave the DNA strand that is complementary to the
20-nucleotide sequence of the crRNA; the RuvC-like domain of Cas9 cleaves the
DNA strand opposite the complementary strand.
 DNA target recognition requires both base pairing to the crRNA sequence and the
presence of a short sequence (PAM) adjacent to the targeted sequence in the DNA
(Jinek et. al.,2012).

12. Source : www.sciencemag.org

13. Mechanism of CRISPR Cas9 mediated Genome Editing
• Genomic Manipulation Requires
－Cas9 protein
－Engineered small-guide RNA with a PAM sequence upstream of target
complementary sequence.
(contains a designed hairpin to mimic CRISPR RNA and trans-acting RNA complex)
• Base-pairing between sgRNA and target DNA causes double stranded breaks
due to endonuclease activity of Cas9.

14. Source : Jinek et al, 2014

15. Improving Cas9 target Recognition Fidelity
(a) A pair of offset Cas9
nickases. The D10A
mutation inactivates the
RuvC endonuclease domain
(Ran et al., 2013)
(b) Cas9-FokI fusion
proteins.
L. Bortesi, R. Fischer / Biotechnology Advances 33 (2015) 41–52

16. APPLICATIONS
• Systematic analysis of gene functions in mammalian cells. (DOI: 10.1126/science.1258096)
• An effective technique that will allow scientists to adequately edit genes to
cure diseases.
-Sickle cell anemia
• Ability to correct genetic mutations responsible for inherited disorders(Wu et al. 2014)
－A dominant mutation in the Crygc gene responsible for cataracts was successfully corrected in mice
• Precise reproduction of tumor-associated chromosomal translocations
(meyerson et al, 2014)
• In Plant Breeding , for eliminating genes that negatively affect food quality and
confer pathogen susceptibility.
(MLO homoalleles in wheat) (Wang et al., 2014)

17. Beyond Genome Editing
• The ectopic regulation of gene expression (Gilbert et al., 2013)
－Inactive Cas9 fused with TAL activation domain in Nicotiana used to regulate gene
expression.
• Inactive Cas9 also used to deliver specific cargos to targeted genomic
locations.(Anton et al., 2014)
L. Bortesi, R. Fischer / Biotechnology Advances 33 (2015) 41–52

18. Continued..........
• RNA Silencing System
L. Bortesi, R. Fischer / Biotechnology Advances 33 (2015) 41–52
• The Type III-B CRISPR-Cas system is composed of nucleases that
form the so-called Cmr complex (yellow) and represents a unique RNA
silencing system.

19. Future Directions
• Alternative PAM sequences can be exploited (Jinek et al., 2015).
• Protein engineering to modify Cas9.
• Methods for efficient delivery and expression of CRISPR-Cas system
need to be optimised.
• Strategies for shifting the balance away from NHEJ-mediated indel
mutations and toward HDR-driven alterations.

21. “There is craze of CRISPR. No other technology have evolved and
talked in previous three years as CRISPR.”
Harvey, Professor Of MIT.
Thank you.......