This document summarizes Jai Kishan's presentation on gene manipulation techniques. It discusses conventional plant breeding, mutation breeding, transgenic plants, and several precise gene editing techniques like ZFN, TALENs, and CRISPR-Cas9. CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by recognizing foreign DNA. The document then reviews the history and components of the CRISPR-Cas system and its applications in cancer therapy, viral diseases, bacterial infections, and genetic disorders. It also discusses challenges like delivery methods, mosaicism, immune responses, and off-target mutations. Key references on the discovery of CRISPR and its use in agriculture and as a genetic manipulation tool are presented.

1. Presented By: Jai Kishan
Roll no : 27191978404
Department : Biotechnology
Batch : 2019-2021

2.  Genes, the fundamental code of life, are written in DNA. Before DNA
was even discovered, people use to manipulate genes via selective
breeding but with time gene manipulation is getting advance and
better with time.
 Conventional Plant Breeding
 Mutation Breeding
 Transgenic Plants
 Antisense Technology
 RNAi Technology
 Zinc Finger Nuclease(ZFN)
 Transcription Activator-
Like endonuclease(TALEN)
 CRISPR-Cas

3.  CRISPR Cas systems are highly diverse adaptive immune system used by most
archae(87%) and bacteria(45%) to protect themselves from invading viruses and
plasmids.
 This system allows cell to recognize and distinguish between ‘foreign’ DNA from ‘Self’
DNA.
 CRISPR stands for CLUSTERED REGULARLY INTERSPACED SHORT PALENDROMIC
REPEATS. It consist of:
 CRISPR locus consist of A-T rich leader sequence(500bp) which carry promoter and
signals for CRISPR System adaptation for transcription of Cr-RNA , Cas Genes and
CRISPR Array.
 Cas genes are the backbone of CRISPR Cas system responsible for silencing and
cleavage of alien nucleotide.
 CRISPR array consist of spacer-repeats sequences which form an array, allows us to
detect foreign nucleic acid along with cas proteins. Repeater sequence is of 28-37bp
approx and spacer size may vary from species to species.

4. Year Work Reference
1987
They while working on alkaline phosphatase, they
discovered
CRISPR
Ishino.et.al,1987
2002 Term CRISPR coined for first time Jensen et.al, 2002
2005 PAM and Cas9 sequence were discovered Bolotin.et.al, 2005
2007 Experimental confirmation that
CRISPR-Cas System provide
Acquired Immunity to prokaryotes
Barrangou et.al, 2007
2008 Found out, CRISPER RNAs are responsible
for antiviral defences
brouns.et.al, 2008
2010 It was found that Cas9 cuts specifically at
PAM sequence
garneau et.al, 2010
2020 Emmanuelle Charpentier and Jennifer A. Doudna
got nobel prize on developement on
CRISPR-Cas System

10. Many people have moral and religious objections to
the use of human embryos for research.
It only to prevent genetic as well as non genetic
diseases.
Use of genetic tools and using or exploiting animals
for our use is working against
If humans would use it for enhancement then no
competition would be there for.
If a small mutation is added one family it will move
in whole generation this can be used as a weapon for
mass destruction among individuals.

11. Increasing yield
Improving Quality
Biotic and Abiotic Stress Resistant
Speeding Hybrid Breeding
Antiviral Plant Breeding
Epigenetic Modification
Transcriptional regulation
Gene Localization
Food and Industrial Applications

12.  Cancer Therapy- CRISPR can be used to dissect the diverse genetic and epigenetic
factors that are involved in cancer and tumor genesis. Through HDR- or NHEJ-mediated
silencing or knock-in of oncogenes and tumour suppressor genes.
 Viral Diseases- CRISPR systems can be employed to combat viral diseases by
disrupting the viral replication mechanisms and restoring the infected cell to normality.
 Bacterial Infections- The emergence of antimicrobial-resistant bacteria is one of
the most concerning aspects for public health specialists nowadays. Misuse and overuse
of antibiotics have led to an increasing number of multidrug-resistant strains , urging the
need for new ways to fight back bacterial infections.
 Genetic Disorders- The correction of diseases arising from genetic aberrations is
one of the most obvious use cases for CRISPR methodologies, due to its ability to produce
specific changes in the genome.

13. Encapsulation
Mosaicism
Stability
Immune Response
Off-Target Mutation
Targeted Delivery

14.  Yoshizumi Ishino et al., 1987 “Nucleotide Sequence of the iap Gene, Responsible for
Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the
Gene Product”
 Alexandre Loureiro et al.,2019 “CRISPR-Cas: Converting A Bacterial Defence
Mechanism into A State-of-the-Art Genetic Manipulation Tool”
 Kunling Chen et al., 2019 “CRISPR/Cas Genome Editing and Precision Plant Breeding in
Agriculture”
 Anaïs Le Rhun et al.,2019 “CRISPR-Cas in Streptococcus pyogenes”
 Fuguo Jiang and Jennifer A. Doudna,2017 “CRISPR–Cas9 Structures and Mechanisms”
 Xingyu Jiang et al.,2019 “Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy

15. THANKS