CRISPR-Cas9 is a bacterial defense mechanism that has been adapted for gene editing, allowing scientists to modify genomes with high efficiency and simplicity. It uses a cas protein complex to identify and cut specific DNA sequences, facilitating gene knockouts and the introduction of new genes. While CRISPR-Cas9 offers advantages such as ease of design and the ability to introduce multiple mutations, it also presents challenges like off-site effects and mosaicism in modified organisms.

1. CRISPR-CAS 9
Hemant Jain and Bhakt singh
keneddy cherukula .. … . , ..

2. What is CRISPR
1. It is a bacterial defence mechanism
2. First discovered in ecoil in 1980s

4. Cluster regularly interspaced short
palindrome repeat

5. Clustered regularly interspaced short
palindromic repeats

6. • Spacer dna – ???
• It was found in 2000 that spacer dna matches perfectly with
bacteriophage dna
• Also the genes associated with crispr gene was discovered i.e CAS
genes

7. CAS genes produces cas protien complex
• This CAS complex has 2 parts
1. hilecase part
2. nucleases part
Therefore it acted
As immune system
Of bacteria.

8. In case when viral dna enters the host cell
• There will be 3 scenarios
Scenario 1

9. Scenario 2
The CRISPR gene
would transcript and
translate Toform CAS
protien complex
That denaturated
the viral DNA by
identifying by the
copy of host RNA as
a template for
recognition

10. Scenario 3
• If the viral DNA dose not
Matches with the
interspaced DNA then a
different class of CAS
protien complex would be
produced that will break the
viral DNA and incorporate it
as an interspaced DNA
among the palindromes .
• Therefore these interspaced
DNA acts as the history of
old infection and hence
developes further immunity
.

11. EUREKA
• Realizing this fact the scientist could now use it to activate or
incorporate new genes in the host

12. • Scientist Jennifer
doudna and
Emmanuelle
Charpentier
identified CAS 9 in
their labs while
working on
steptococcus
pyogenes

13. CAS 9
• It has crRNA
(SPACER
DNA ) and
trRNA that
holds crRNA
in place

14. • The scientists
modifiedthis system
by using cas 9
protien and use the
host cell’s RNA and
by connecting it to
tracrRNA i.e.
trcrRNA-crRNA
chimera also called
grRNA.

15. • Due to this
the CAS 9
detects the
required DNA
sequence by
using the
host’s own
RNA as a
template and
cuts it out
• After that
DNA
reparation
takes place

16. The RNA in CAS 9 recognises the DNA
sequence and cleaves it

17. After cleaving the DNA reparation/ ligation
takes place

18. Hence a knockout could be produced

19. By this system a new gene could be induced
at the place of deleted gene

21. Advantages of CRISPR/CAS over ZNF and
TALEN mutagenesis stratagies
• 1. Target design simplicity. Because the target specificity relies on
ribonucleotide complex formation and not protein/DNA recognition,
gRNAs can be designed readily and cheaply to target nearly any
sequence in the genome specifically.
• 2. Efficiency. The system is super-efficient. Modifications can be
introduced by directly injecting RNAs encoding the Cas protein and
gRNA into developing mouse embryos. This eliminates the long and
laborious processes of transfecting and selecting mouse ES cells that
are required to create targeted mutant mice using classical
homologous recombination techniques.

22. • 3. Multiplexed mutations. Mutations can be introduced in multiple
genes at the same time by injecting them with multiple gRNAs

23. Disadvantages of CRISPR-CAS 9
• 1. Off-site effects. Mutation introduced at non-specific loci with similar, but not
identical, homology to the target sites are one of the most important
complication of these technologies. These can be difficult to identify and require
scanning the genome for mutations at sites with sequence similarity to the gRNA
target sequence.
• 2. Mosaicism. Mice with a mutant allele in only some of their cells can be
produced , because the nucleases may not necessarily cut the DNA at the one cell
stage of embryonic development.
• 3. Multiple alleles. Healing of the nuclease cleavage site by non-homologous end
joining can produce cohorts of mice with different mutations from the same
targeting constructs, requiring genome sequencing to verify the nature and
position of the specific mutation. The production of mice with mosaics of
multiple mutations, also, is possible, and breeding may be required to segregate
and isolate mice that carry single mutations. The production of mice with
multiple variants also creates phenotyping bottlenecks.

24. CRISPR has
Many applications

25. CRISPR in medicine HIV/AIDS

26. Other medical applications
Muscular Dystrophy
Cancer
And Many more..

27. CRISPR-Cas9 in functional genomic screening
• Functional genomic screening is largely used for identifying the
essential genes for a specific cellular process.

28. CRISPR-Cas9 in correction of genetic disorders
• One of the most exciting applications of the CRISPR-Cas9 is the
possibility of curing genetic diseases.
• Eg . Could event cataract by inducing mutation in CYRC gene
• Similarly could also cure cystic
fibrosis by targeting multiple
genes

29. CRISPR-Cas9 in the treatment of infectious diseases
• Considering that the CRISPR-Cas system originally functions as an
antiviral adaptive immune system in bacteria, this system could be
used for treating infectious diseases by eradicating pathogen
genomes from infected individuals.

30. CRISPR-Cas9 in the generation of animal models
• gene targeting based on homologous recombination and embryonic
stem cells has been used as the typical approach for animal genome
modification, which has played indispensable roles in making a causal
link between genomic mutations and phenotypes during
development and in disease. However, gene targeting has limited
applications in some organisms due to time-consuming procedures
and the lack of available embryonic stem cells