CRISPR/Cas9 is a powerful genome editing tool that allows genetic material to be added, altered or removed at specific locations in the genome. It involves a bacterial adaptive immune system where CRISPR sequences and Cas genes work together. The Cas9 protein uses a guide RNA to introduce double stranded breaks at targeted DNA sequences. This enables precise genome editing through non-homologous end joining or homology directed repair. CRISPR/Cas9 provides a simple and accurate way to modify genes for applications in research, medicine, agriculture and more. While it holds great promise, there are also limitations and concerns regarding off-target effects that researchers continue working to address.

1. 'CRISPR/Castechnology'
ANJU H.
MPhil Advanced Botany
UNIVERSITYOF KERALA, KARYAVATTOM

2. Genetic Engineering
“The deliberate modification of the characteristics of
an organism by manipulating its genetic material.”
•Research
•Medicine(Protein/Enzyme production)
•Agriculture(Crops)
•Industrial Biotechnology (Biofuel production)
•Entertainment

3. What are genome editing ?
• Genome editing (also called gene editing) is a group of technologies
that give scientists the ability to change an organism's DNA. These
technologies allow genetic material to be added, removed, or altered
at particular locations in the genome.
• Several approaches to genome editing have been developed.
Zinc-finger nucleases
TALENs
CRISPRs

4. Proteinbased
RNA based
Zinc Fingers
TALEN
CRISPR

5. Why is CRISPR/Cas9 better?
Accuracy
Simplicity
Speed

9. TIMELINE

10. 2012 Proposal CRISPR for Genome editing (Jinek, Doudna, Charpentieret al.)
2013 CRISPR used for genome editing in eukaryotic cells (Zhang et al.)
2014 Crystal structure of Cas9 gRNA complex (Nishimasu, Zhang et al.)

11. - Clustered Regularly Interspaced Palindromic Repeats
• Consists of short, repetitive DNA sequences palindrome (20-40 bp ) Interspaced
with non repetitive sequences( spacers)
• Spacers were identical to many sequences present in exogenous mobile genetic
elements like plasmids, transposons, and bacteriophages
• CRISPR arrays were often associated with a set of Cas genes; encodes Cas proteins
• Cas genes had sequence similarity to endonuclease and helicase families or genes
encoding other nucleic acid binding proteins
• Trans activating RNA coding gene
Repetitive DNA sequence palindrome
spacers
Cas genes
Trans activating RNA coding gene

12. Cas 9 (CRISPR associated protein)
 RNA guided DNA endonuclease enzyme
 Involves in type ll CRISPR mechanism
6 domains
o REC I responsible for binding guide RNA
o REC II not yet well understood
o Bridge Helics (arginine rich) is crucial for initialising clevage activity
upon binding of target DNA
o PAM interacting domain: responsible for initialising binding to target
DNA
o HNH RuvC domains: nuclease domain that cut single stranded
DNA. They are highly homologus to HNH and RuvC domains found in
other proteins. RuvC - cleaves non-targetDNA strand HNH - cleaves-
targetstrandof DNA
Cas9
(S. pyogenes)
REC I
REC II
PAM interacting domain
HNH RuvC

13. CRISPR-Cas-Adaptive immune system
(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 DNAby
Cas nuclease cleavage

14. PAM (protospacer adjacent
motif)-specificity of crRNA for
the target is increased by
specific nucleotide sequences
adjacent to the target

15. Developeda simplifiedtwo-component system by combining
tracrRNA and crRNA into a single synthetic single guide RNA (sgRNA).

17. Types of CRISPR-Cas systems

23. Non- Homologous
End Joining
Non- Homologous
End Joining
Homology Directed
Repair
Homology Directed
Repair
Non-
Homologous
End Joining
Homology
Directed
Repair

24. Some pitfall in this technology
Limited PAM sequence
Off target intels

25. Enhancing specificity of CRISPR/Cas9 technology
1. NickaseActivity

26. Enhancing specificity of CRISPR/Cas9 technology
1.NickaseActivity
2.FokI-Fusion

27. Enhancing specificity of CRISPR/Cas9 technology
1.Nickase Activity
2.FokI-Fusion
3.Improvedtargetrecognition

31. Applications of CRISPR/Cas9
Delivery of desired gene
Knock out /knock in
Gene activation and repression
Genome visualization
Genotyping of polymorphism
Disease model study
Biomedicine development
Provides broad spectrum
resistance

35. GenomeeditingusingCRISPR/Cas9–targetedmutagenesis:An
opportunityfor yield improvementsof crop plantsgrownunder
environmental stresses. ( MostafaA et al. 2018)
1. CRISPR/Cas9 applications for plant disease resistance
-In Duncan grapefruit (Citrus paradisi) GE mutagenesis, using the CRISPR/Cas9
system, of the effector binding elements (EBEs) in the promoter region of two
alleles of the canker susceptibility lateral organ boundaries 1 (CsLOB1) transcription
factor (TF)
-CRISPR/Cas9-induced modification of the EBEs in the promoter of the S/CsLOB1
gene enhances disease resistance in Wanjincheng orange (Citrus sinensis Osbeck)
against X. citri subspecies.
2. CRISPR/Cas9 applications for improvement of abiotic stress
resistance in crop plants
- importance of the SlMAPK3 gene for drought tolerance in tomato
plants

36. Seamlessgene correction of 𝜷-thalassemia mutations in
patient-specificiPSCs using CRISPR/Cas9and
piggyback.(Xie et al. 2014)
• caused by mutations in the human hemoglobin beta (HBB) gene
• CRISPR/Cas9 technology, combined with the piggyback transposon to
efficiently correct the HBB mutations in patient-derived iPSCs without
leaving any residual footprint.
• When differentiated into erythroblasts using a monolayer culture,
gene-corrected iPSCs restored expression of HBB compared to the
parental iPSCs line.

37. Gene-edited CRISPR mushroom escapes US
regulation(Emily Waltz, Nature Issue-7599)
• Polyphenol oxidase (PPO) causes browning of mushrooms during storage
• CRISPR to introduce mutations to 1 out of 6 PPOgenes
• 30% reduced activity
• Prolonged storage time

38. Using CRISPR/Cas9 genome editing in tomato to
create a gibberellin-responsive dominant dwarf
DELLA allele.(Tomlinson L et al.2018)
• The tomato PROCERA gene encodes a DELLA protein, and
loss‐of‐function mutations derepress growth.
• CRISPR/Cas9 and a single guide RNAs (sgRNA) to target mutations to
the PROCERA DELLA domain, and recovered several loss‐of‐function
mutations and a dominant dwarf mutation that carries a deletion of
one amino acid in the DELLA domain.
• This dominant dwarf PROCERA allele retains partial responsiveness to
exogenously applied gibberellin.

39. CRISPR-edited rice plants produce major boost
in grain yield(Zhu et al.2018)
• CRISPR/Cas9 gene-editing technology to develop a variety of rice that
produces 25-31 percent more grain and would have been virtually
impossible to create through traditional breeding methods.
• Made mutations to 13 genes associated with the phytohormone
abscisic acid, known to play roles in plant stress tolerance and
suppression of growth.

41. THANK YOU