This document provides an overview of CRISPR-Cas as an advanced and efficient tool for genome modification. It describes how CRISPR-Cas systems incorporate DNA from invading viruses or plasmids and use RNA-guided Cas nucleases to cleave matching sequences in foreign DNA. The two main components required for genome editing are Cas9 nuclease and a guide RNA. By altering the guide RNA sequence, Cas9 can be directed to cleave any desired DNA target. The document discusses applications of CRISPR-Cas in genome editing, gene regulation, molecular barcoding, and potential future uses in medicine and biotechnology.

1. CRISPR-CAS: AN
ADVANCED AND
EFFICIENT TOOL FOR
GENOME MODIFICATION
by
Avinash Tiwari
M. Tech 1st year, 2nd sem.
201710209010002

2. Introduction

3. • Every bacterium has their own resistance mechanism against the
exposure of foreign genetic material
• The CRISPER (clustered regularly interspaced short palindromic
repeats) provide these defensive belongings
• frequently found in archaea and typically in bacteria, although this
system is firstly found in E. coli

4. • virus attack on the bacteria the insert their DNA in the bacteria and
hijack the bacterial machinery and make their copy and spread
• CRISPER is like cut and paste is ligated in bacterial genome in a
specific area
• incorporated DNA segment of virus transcribed in to RNA which is
associated with a cas nucleases enzymes which snip the virus DNA
and make the disable

5. Mechanism

7. How it work

8. • CRISPR system incorporates sequences from invading DNA between
CRISPR repeat sequences encoded as arrays within the host genome
• Transcripts from the CRISPR repeat arrays are processed into CRISPR
RNAs (crRNAs) having a variable sequence transcribed from the invading
DNA, known as the “protospacer” sequence, as well as part of the
CRISPR repeat
• The protospacer encoded portion of the crRNA directs cas9 to cleave
complementary target DNA sequences. CRISPR system from
Streptococcus pyogenes has been adapted for inducing sequence-
specific DSBs and targeted genome editing

9. • Two components must be introduced and/or expressed in cells or an
organism to perform genome editing:
1. the cas9 nuclease
2. a ‘guide RNA’ (gRNA),
• consisting of a fusion of a crRNA and a constant tracrRNA.20
nucleotides at the 5′ end of the gRNA direct cas9 to a specific target
DNA site using standard RNA-DNA complementarity base pairing rules
• Thus, with this system, cas9 nuclease activity can be directed to any
DNA sequence of the form N20 -NGG simply by altering the first 20
nucleotide of the gRNA to correspond to the target DNA sequence

11. Schematic diagram of a CRISPR/Cas single
plasmid format

12. Application

13. • genome editing
• completely change the genome of any eukaryotic organism
• endogenous gene expression •
• endogenous transcript and encoding a bacterial lipoprotein (BLP)
• gene regulation in the pathogenesis
• transformed genome editing and biomedicine
• applied as genetic recorders
• molecular barcoding and cellular lineage tracing
• provides a cellular memory of biological activity

14. Future Aspects

15. • Pharmaceutical companies may develop the CRISPR based drug for
treat the Heart disease, blood disorders and blindness
• It will be possible in couple of decades , it may be possible to treat
cancer , AIDS through a pill or injections
• CRISPR-cas may be used for boosting human immune intelligence

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