CRISPR Cas9 Kshirsagar Ravindra molecular biology

CRISPR Cas9 Kshirsagar Ravindra molecular biology Clustered Regularly Interspaced Short Palindromic Repeats

1. By
Dr.Ravindra V. Kshirsagar
Modern College Ganeshkhind Pune
CRISPR-Cas9
Clustered Regularly Interspaced Short Palindromic Repeats

2. What is CRISPR-Cas9?
ANS:
CRISPR stands for Clustered Regularly Interspaced Short
Palindromic Repeats, a family of DNA sequences found in the
genomes of prokaryotic organisms like bacteria and archaea.
These sequences serve as a part of the immune system for these
organisms, allowing them to recognize and defend against viral
infections

3. Cas9 (CRISPR-associated protein 9) is an enzyme that acts as
molecular scissors, cutting DNA at specific locations guided
by RNA sequences.

4. CRISPR-Cas9 is a revolutionary gene-editing technology that allows scientists to modify DNA with
precision. It consists of two main components: CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats), which acts as a genetic memory system, and Cas9, an enzyme that cuts DNA at specific locations.
Originally discovered as a bacterial defense mechanism against viruses, CRISPR-Cas9 has been adapted for
genetic engineering. Scientists use a guide RNA (gRNA) to direct Cas9 to a specific DNA sequence, where it
makes a cut. This allows researchers to edit, delete, or replace genes, making it a powerful tool for medicine,
agriculture, and biotechnology.
Applications of CRISPR-Cas9 include:
Medical research: Potential treatments for genetic disorders like sickle cell anemia and cystic fibrosis.
Agriculture: Developing disease-resistant crops and improving food production.
Biotechnology: Creating genetically modified organisms for research and industry

5. Mechanism of CRISPR-Cas9: Step-by-Step
. Target Recognition (Guide RNA)
Scientists design a guide RNA (gRNA) that is complementary to a specific DNA sequence
in the genome.
The gRNA forms a complex with the Cas9 enzyme.
The guide RNA leads Cas9 to the target DNA sequence by base-pairing.

6. 2. DNA Binding
Cas9 binds to the target DNA, but only if it’s adjacent to a short sequence called the PAM (Protospacer
Adjacent Motif), which is usually NGG in the case of Cas9 from Streptococcus pyogenes.
3. DNA Cleavage
Cas9 induces a double-strand break (DSB) at the target site by cutting both DNA strands.
This break occurs precisely at the location dictated by the guide RNA.

7. 4. DNA Repair (by the Cell)
After the DNA is cut, the cell tries to repair it in one of two main ways:
•Non-Homologous End Joining (NHEJ):
•An error-prone process.
•Often results in insertions or deletions (indels).
•Can disrupt or "knock out" a gene.
•Homology-Directed Repair (HDR):
•A precise process if a template DNA is provided.
•Can be used to insert, correct, or replace specific DNA sequences.

8. Applications of CRISPR-Cas9
Gene therapy (e.g., treating genetic diseases like sickle cell
anemia).
Agriculture (e.g., engineering crops with desirable traits).
Functional genomics (e.g., studying gene functions by knocking
them out).
Drug development and disease modeling.

9. CRISPR-Cas9