CRISPR is a groundbreaking genome editing tool derived from bacterial immune systems, enabling precise modifications of DNA through components like guide RNA and Cas9 proteins. Its development has evolved since its initial discovery in 1987, culminating in practical applications for gene editing, disease prevention, and agricultural improvements. While it offers significant advantages such as precision and versatility, ethical concerns and potential off-target effects present challenges to its widespread adoption.

1. CRISPR
A Genome Editing Tool

3. INTRODUCTION:
Clustered Regularly Interspaced Short Palindromic Repeats”
New gene editing technology with the potential to
revolutionize genetic engineering and the biotechnology
industry.
Use to understanding, characterizing, and controlling DNA.
 Based on bacterial immune system.
 Single solution to many problems
CRISPR are found in approximately 50% of sequenced bacterial
genome and nearly 90% of sequenced Archaea.

4. HISTORY:
 1987 Researcher youshizmi Ishano find CRISPR sequence in E.coli but they don’t
characterized their functions.
 2000 CRISPR sequence were found by moica to be common in other microbes.
2002 Mr. Jansen Coined CRISPR name, defined, signature cas gene.
2007 First experimental evidence for CRISPR adaptive immunity.
2008 Spacer sequences are transcribed into guide RNAs
2010 Discovered that Cas9 can cleaves target DNA
2011 Discovery of tracrRNA for Cas9 system
2011 Discovered that CRISPR systems can function heterologously in other species
2012 Idea of using CRISPR- Cas9 as a genomic engineering tool, was published by
Jennifer and Emmanuelle.
 2013 First demonstration of Cas9 genome engineering in Eukaryotic cell.
2014 First CRISPR patent was granted to Feng Zheng.

5. Components of CRISPR
1. Guide RNA (gRNA)
tracrRNA
crRNA
2.Protospacer associated Motif (PAM)
3.CRISPR-associated endonuclease
protein(Cas enzymes)

7. Cas9 Protein
• S. pyogenes Cas9 is a large multi-domain and
multifunctional DNA endonuclease. It cut
dsDNA 3 bp upstream of the PAM through. Its
two distinct nuclease domains:
1. An HNH-like nuclease domain that cleaves
the DNA strand complementary to the guide
RNA sequence (target strand)
2. 2nd is RuvC-like nuclease domain responsible
for cleaving the DNA strand opposite the
complementary strand (non-target strand).
In addition to its critical role in CRISPR
interference, Cas9 also participates in crRNA
maturation and spacer acquisition.

8. Guide RNA Variants
•Truncated Guide RNAs
(truRNA)
•Ribozyme-gRNA-Ribozyme
(RGR)
•Polycistronic tRNA-gRNA
(PTG/Cas9)

9. The Cas9 Nuclease Variants
•The Native Cas9
•The Cas9 Nickase
•The Inactive dCas9
•Dimeric RNA-Guided
FokI Nucleases
(RFNs)

10. The CRISPR immune response three steps:
1. Adaptation: When DNA from a virus invades the bacteria, the viral DNA
is processed into short segments and is made into a new spacer between
the repeats. These will serve as genetic memory of previous infections.
2. Production of CRISPR RNA: The CRISPR sequence undergoes
transcription, including spacers and Cas genes, creating a single-stranded
RNA. The resulting single-stranded RNA is called CRISPR RNA, which
contains copies of the invading viral DNA sequence in its spacers.
3. Targeting : The CRISPR RNAs will identify viral DNA and guide the CRISPR-
associated proteins to them. The protein then cleaves and destroys the
targeted viral material.

11. Mechanism

12. How Does the CRISPR-Cas System Work
Every CRISPR experiment can be
divided into three main steps:
Design the CRISPR sgRNA
 Edit DNA Precisely with CRISPR
Analyze Data from CRISPR
Experiment

14. CRISPR-Cas9 allows to perform the
following
•Gene Knock-Out
•DNA-Free Gene Editing
•Gene Insertions or “Knock-ins”
•Transient Gene Silencing

15. Applications and Future perspectives
Edit crops to be more
nutritious
Tools to stop genetic
diseases
Genome screening
Gene drives
Future perspectives

16. Pros
• Reverse respectively all the
mutations
• Fast then others
• Utilize in many different
species
• Greater precision
• Excellent ability to target
any genomic region
Cons
•Off target effects like
Secondary unwanted
mutation
• Mosaic effects
• Ethical
•Social effects on the
society
•It Is Not Always
Efficient

17. The End