This document discusses genome editing using the CRISPR-Cas9 system. It begins by introducing three main genome editing technologies - zinc-finger nucleases, TALENs, and the CRISPR-Cas9 system. It then describes the key events in the discovery of CRISPR-Cas9, including its origins as a bacterial defense system. The document outlines the main components of the CRISPR-Cas9 system, including crRNA, tracrRNA, sgRNA, and Cas9. It also summarizes the two main steps in genome editing using CRISPR-Cas9 - knocking out genes and DNA repair. The document concludes by discussing opportunities for applying CRISPR-Cas9 technology across various

1. GENOME EDITING BY :

2. INTRODUCTION
 A number of genome editing technologies have emerged :
zinc-finger nucleases (ZFNs), transcription activator–like
effector nucleases (TALENs) and the RNA-guided CRISPR-
Cas nuclease system.
 The first two technologies use a strategy of tethering
endonuclease catalytic domains to modular DNA-binding
proteins for inducing targeted DNA double-stranded breaks
(DSBs) at specific genomic loci.
 By contrast, Cas9 is a nuclease guided by small RNAs through
Watson-Crick base pairing with target DNA representing a
system that is markedly easier to design, highly specific,
efficient and well-suited for high-throughput and multiplexed
gene editing for a variety of cell types and organisms.

3. DISCOVERY
 The first description of what would later be called
CRISPR is from Osaka University researcher
Yoshizumi Ishino and his colleagues in 1987.
 Jennifer Daudna and Emmanuelle Charpentier re-
engineered the Cas9 endonuclease into a more
manageable two-component system by fusing the
two RNA molecules into a "single-guide RNA" that,
when combined with Cas9, could find and cut the
DNA target specified by the guide RNA. By
manipulating the nucleotide sequence of the guide
RNA, the artificial Cas9 system could be
programmed to target any DNA sequence for
cleavage.

4. Emmanuelle charpentier (L) and Jennifer doudna (R)

5. CRISPR- CAS SYSTEM AS A DEFENCE
MECHANISM OF BACTERIA AND
ARCHEA

19. GENOME EDITING WITH CRISPR/CAS9
TECHNOLOGY

20. KEY COMPONENTS
crRNA : Contains the guide RNA that locates the
correct section of host DNA along with a region that binds
to tracrRNA (generally in a hairpin loop form) forming an
active complex.
tracrRNA : Binds to crRNA and forms an active
complex.
sgRNA : Single guide RNAs are a combined RNA
consisting of a tracrRNA and a crRNA
Cas9 : Protein whose active form is able to modify
DNA. Many variants exist with differing functions (i.e.
single strand nicking, double strand break, DNA binding)
due to Cas9's DNA site recognition function.

21. MAINLY TWO STEPS ARE INVOLVED
Gene knocking
out
DNA Repair

37. CRISPR-CAS9: THE FUTURE OF GENE EDITING
AND GENETIC COUNSELING!!

38. THESE ARE THE OCEAN OF OPPORTUNITIES THAT CAN BE
EXPLORED WITH THE HELP OF THIS..!!
 Fighting cancer
 Extracting HIV
 Making diseases self destruct
 Improving IVF
 Eliminating malaria
 Protecting plants
 Producing food
 Creating biofuel
 Reviving extinct mammals

39. IN AGRICULTURE
 Can be used to create high degree of genetic variability
at precise locus in the genome of the crop plants.
 Potential tool for multiplexed reverse and forward
genetic study.
 Precise transgene integration at specific loci.
 Developing biotic and abiotic resistant traits in crop
plants.
 Potential tool for developing virus resistant crop
varieties.
 Can be used to eradicate unwanted species like
herbicide resistant weeds, insect pest.
 Potential tool for improving polyploid crops like potato
and wheat.

40. CASE STUDY

41. OVERVIEW
 Sequential transformation method for gene
targeting in Arabidopsis was adopted.
 Parental lines expressing the bacterial
endonuclease Cas9 from the egg cell- and early
embryospecific DD45 gene promoter can improve
the frequency of single-guide RNA-targeted gene
knock-ins and sequence replacements via
homologous recombination at several endogenous
sites in the Arabidopsis genome.
 PCR approach was used to identify the heritability
of the gene.

42. METHODS & MATERIALS
 Gene accession numbers: ROS1, At2g36490; DME,
At5g04560; GL2, At1g79840.
 Plant materials and growth condition.
Arabidopsis thaliana accession Col-0
MS media used, at 22 °C, 1% sucrose. hygromycin (25 mg/L) containing MS plates used to
selection
 Plasmid construction:The optimized coding sequence of
hSpCas9 (CRISPR/Cas9) plasmids for GL2 GT were constructed in pCambia1300. For all-in-
one GT constructs, donor sequence was added to the published CRISPR/Cas9 constructs. For
GT constructs for the sequential transformation strategy, AtU6 promoter-driven sgRNA and
donor sequence were constructed in pCambia330.
 DNA analysis.
 RNA analysis.
 Detection of GFP fluorescence and Luc
luminescence.
 DNA methylation analysis.

43. RESULTS AND CONCLUSION
 Precise knock-ins, generating ROS1-GFP, ROS1-Luc,
DME-GFP, and GFP-DME fusions were achieved.
 Only DD45 promoter-driven Cas9 lines yielded heritable
GT
 HDR may be more efficient in egg cells and/or early
embryos than in other germline tissues (e.g., pollen and
shoot apical meristem).
 Five GFP-DME heterozygous T2 plants showed
segregation from the Cas9 transgene , indicating that
heritable knock-in occurred in T1 plants.
 The GT efficiency by our method was 5.3% for DME
P1633A and was higher for other knock-ins or gene
replacement.
 Results show that GT events were not related to T-DNA
copy numbers of Cas9 or of the HDR donor transgene