ALL ABOUT CAS9 PROTEIN INTRO, HISTORY,HOW IT WORKS ,ETHICAL CONCERNS

1. CRISPR/Cas technology-the gene editing
tool
presented by:
uwesh kazi ,
SINGH SHRUTI
verma chandni (m pharm ,1st year)
Guided by : professor krutika sawant mam

2. OUTLINE
• INTRODUCTION TO GENETIC ENGINEERING
• CRISPR- BRIEF
INTRODUCTION,DEFINATION,HISTORY
• COMPONENTS OF CAS9 TECHNOLOGY
• MECHANISM OF WORKING,VARIATION
• APPLICATIONS
• REGULATION-MOST IMPORTANT
• RECENT ADVANCES
• REFERENCES

3. • GENETIC ENGINEERING /TRANSGENICS/
GENE TRANSFER
TECHNIQUES ARE AS FOLLOWS :
RECOMBINANT DNA
ELECTRO AND CHEMICAL PORATION
MICROINJECTION
BIOBALLISTICS METHOD.
PCR

4. CRISPR- CLUSTERED REGULARLY INTERSPACED
SHORT PALINDROMIC REPEATS

5. HISTORY OF CRISPR
• 1987 1st report on repetitive sequences (CRISPR, Ishano et al.)
• 2000 CRISPR present throughout prokaryotes (Mojica et al.)
• 2005 Foreign elements, proposed immunity function (Mojica et al.)
• 1987 1st report on CRISPR (Ishano et al.)
• 2000 CRISPR present throughout prokaryotes (Mojica et al.)
• 2005 Foreign elements, proposed immunity function (Mojica et al.)
• 2010 Cas9 is guided by spacer and induces DSB in target (Garneau et al.)
• 2011 Heterologous expression of CRISPR type II (Sapranauskas et al.)
• 2012 Proposal CRISPR for Genome editing (Jinek, Doudna, Charpentier et
al.)
• 2013 CRISPR used for genome editing in eukaryotic cells (Zhang et al.)
• 2014 Crystal structure of Cas9 gRNA complex (Nishimasu, Zhang et al.)

6. The CRISPR – Cas system is a genome editing technique
that allows to alter the genetic code of any given
organism.
• CRISPRs are short DNA sequences, each about 30 bases long ,
read similarly backwards and forwards and repeat every 35
bases or so.
• Cas(CRISPR associated) protein is able to cut the invading
phage DNA into small fragments, which are then integrated
into the CRISPR array as a spacer .
• A CRISPR ARRAY is composed of series of repeats interspaced
by spacer sequences acquired from invading genomes.

7. KEY COMPONENTS
crRNA - Contains the guide RNA that locates the
correct section of host DNA along with a region that
binds to tracrRNA forming an active complex.
tracrRNA – Binds to crRNA and forms an active
complex.
sgRNA – single guide RNAs are combined RNA
consisting of a tracrRNA and at least one crRNA.
Cas9 – protein whose active form is able to modify
DNA. Many variants exits with different functions
due to Cas9’s DNA site recognition function

8. STRUCTURE
Cas9 has six domains.
The REC 1 domain is the largest and
it is responsible for binding guide
RNA and role of REC 2 domain is not
yet well understood.
The arginine-rich bridge helix is for
initiating cleavage activity.
The PAM-interacting domain is
responsible for initiating binding to
target DNA while HNH and RuvC are
nuclease domains that cut single
stranded DNA

9. GUIDE RNA
The guide RNA is engineered
to have a 5’ end that is
complimentary to the target
DNA sequence.
This artificial guide RNA bind
Cas9,induces some
conformational changes and
make it active.
Then both act on target DNA.

19. Different CRISPR-Cas system in
Bacterial Adaptive Immunity
Class 1 Class 2
• Type2(CRISPR-Cas9)
• Type5(CRISPR-Cpf1)
• Employ large single
component Cas9
protein in conjunction
with crRNA ad
tracerRNA
• Type1(CRISPR-Cas3)
• Type3 (CRISPR-Cas10)
• Uses several Cas
proteins and the crRNA

20. MECHANISM
• 1. ADAPTATION
• 2. EXPRESSION AND MATURATION
• 3. INTERFERENCE

22. MECHANISM
Cas9 PROTEIN
Searches for target DNA by binding
with sequences that match its
protospacer adjacent motif(PAM)
sequence
GUIDE RNA
Have a 5’end that is complimentary
to the target DNA sequence.
If the complimentary region and the
target region pair properly,the RuvC
and HNH nuclease domains(of Cas9)
will cut the target DNA.

24. What makes CRISPR system the ideal
genome engineering technology
Key Enabling Attributes To
Become Next Big Drug Class
Broadest Potential To
Modulate Genes
• Ability to address any site in
the genome or foreign
genomes
• Ability to target multiple
DNA sites simultaneously.
• Multifunctional
programmability delete ,
insert or repair genes
• High potency(cleavage
efficiency) and specificity.
• Broad applicability to both
in vivo and ex vivo
applications.
• Simple editing tools(guide
RNA plus protein)allow
unprecedented ability to
scale and optimize at speed
• Potential one time
curvature treatment

25. PROS AND CONS
PROS
• Reverse respectively all
the mutations.
• Faster than others.
• Utilize in many different
species.
• Excellent ability to
target any genomic
region.
CONS
• Off target effects
• Mosaic effects
• Ethical and social
efffects on the society

26. DRAWBACKS,PROBLEMS AND THEIR
SOLUTIONS
MOSAIC PROBLEM
• Safety
• Efficiency : in this unwanted gene sequence form
embryonic stage and causes cancer.
Its solution is adding new CRISPR machinery in
the cell add double standard DNA.
• Wrong attachments:- double stranded DNA is
solution
• Ethical barriers
• Bio weapons

27. Applications of CRISPR in different
Fields:
Microbiology
Biomedicine
Animals
Agriculture
Therapeutic
In vivo

29. MICROBIOLOGY
CRISPR/dCas9-mediated protein imaging.
Fluorescence in-situ hybridisation(FISH)use for
labelling genetic loci.
MECHANISM:- dCas9+yellow/green FP gives
multicolour CRISPR complex which bind with
genetic loci matching sgRNA and labelling done.

30. Why Protein Imaging is done?
Direct visualization of genomic loci facilitates
deep understanding of spatial organisation of
microbial genome and gene expression.
This technique is also effective to:-
• Identify repetitive sequences such as
Isoenzymes and RNA
• Substantially effect biosynthesis of desired
metabolites.

31. Biomedicine
Epstein Barr Virus(EBV)
• CRISPR eradicate viral DNA of EBV
• CRISPR is an Anti-herpes virus removing cancer
causing EBV from tumour cells.
Transplantation
• Gene editing of mismatched humans or even non
human mammals as potential organ donors.
• Editing will reduce risk of immune responses and
rejection when using mismatched organ.

32. Animals
 Cashmere goat
CRISPR/Cas9 system FGF Gene
• Increased number of second hair follicles.
• Enhanced fibre length.
• More cashmere produce
• This change induced at:-
a. Morphological level
b. Genetic level

33. Agriculture
•Potential tool for developing
virus resistance crop variety.
•Can be used to eradicate
herbicide resistance weeds,
insect pest.
•Developing biotic and abiotic
resistance traits in crop
plants.

34. Revolutionary aspects of CRISPR Cas9
• It was stated that- CRISPR is dramatically
accelerating the pace of research in nearly
every biological field.
• CRISPR Cas9 tool use in the Fixing of the
Donor Organ Shortage which one is a
revolution in medical sciences.
• An alternative to Petroleum.
• Designer Pets and Service Animals.

35. Few examples
Sickle cell anemia is a great
example of a disease in which
mutation of a single base
mutation(T to A) could be edited
by CRISPR and the disease cured.
CRISPR/Cas9 successfully
knock out the mildew-resistance
locus(MLO) in wheat to generate
plants resistant to powdery
mildew disease.

36. Continued
Germline manipulation with
CRISPR-Cas9 system in mice
were capable of correcting both
the mutant gene and cataract
phenotype in offspring initially
caused by a one base pair
deletion in exon 3 of
Crygc(crystalin gamma C) gene.

37. Continued
Likewise,in an effort to confirm
that gene editing was at least
possible,cells from rice plants
were transformed with vectors
carrying CRISPR gateway vector
targeting CHLOROPHYLLA
OXYGENASE1(CAO1).
CRISPR-Cas9 can mutate long
terminal repeat(LTR)sequence of
HLV1 in vitro,resulting in removal
of the integrated pro viral DNA
from the host cells with significant
drop in virus expression

38. Rectification of CFTR
Human intestinal stem cells
collected from patients
with cystic fibrosis , the
culprit defective gene
CFTR(cystic fibrosis
transmembrane
regulator)was rectified by
homologous
recombination during
CRISPR-Cas9 genome
editing while the
pluripotency was retained
as demonstrated by
formations of organ like
expansions in cell culture

39. Continued
HepG2 cells expressing hepatitis B virus
(HBV),the introduction of CRISPR-Cas9 system
resulted in both decreased hepatitis B core
antigen expression which provides an
impetus for further research on the
posssibility of CRISPR-Cas9-mediated
hepatitis B prevention.

40. Expanding the Research Applications
for CRISPR
CRISPR/Cas9 technology has been adopted for
many research applications beyond than
genome editing,such as:-
CRIbSPR/Cas9-mediated Chromatin
Immunoprecipitation.
CRISPR Technologies for Transcriptional
Activation and Repression.
Epigenetic editing with CRISPR/Cas9.
Live Imaging of DNA/mRNA with CRISPR/Cas9.

41. THERAPEUTIC APPLICATIONS

45. RECENT ADVANCES

47. CONCLUSION
• The CRISPR/Cas9 genome editing system with its
accelerated development and expanded
applications, is and indispensable tool for precise
and efficient genome editing ,but some related
problems need more attention.
• First, the current knowledge of the CRISPR/Cas9
system at the biochemical and crystal structural
levels is insufficient and requires additional
research, including a deep analysis of the Cas9
protein, one of the main components in the
CRISPR/Cas9 system.

48. CONCLUSION
• The natural variation in Cas9 proteins isolated from
different species might provide new Cas9 proteins
with
higher efficiency and thereby broaden the choices
available for precise genome editing (Ran et al.,
2015) the direct and precise genome editing raises
ethical concerns, such as gene modification of
human germ line cells using the CRISPR/Cas9 system
to create “engineered babies” (Liang et al., 2015),
which initiates arguments and queries among
scientists and the public.

49. CONCLUSION
• In addition, the invention of MCR also creates
intense concern regarding environmental balance
and species safety (Gantz and Bier, 2015). It is urgent
that the government and related social organizations
formulate and enact a series of laws and regulations
to enable the safe and ethical application of the
CRISPR/Cas9 system in basic research and clinics.
• We envisage a bright future in which the
CRISPR/Cas9 system will facilitate revolution and
improvement of genome, RNA, and epigenome
editing.

50. REFERENCES
• RNA-guided genetic silencing systems in bacteria
and archae.Blake Wiedenheft, Samuel H
Sternberg and Jennifer A. Dounda.Nature 2012
• Multiplex Genome Engineering Using
CRISPR/Cas9 Systems . Le Cong et al. Science
2013.
• Orthogonal Cas9 proteins for RNA-guided gene
regulation and editing.Esvelt et al.Nature
Methods 2013.
• A Programmable Dual RNA-Guided DNA
Endonuclease in Adaptive Bacterial
Immunity.Jinek et al.Science 2012

51. REFERENCES
• RNA-guided editing of bacterial genomes using
CRISPR-Cas systems.Jiang et al.Nature Biotech
2013
• Dynamic Imaging of Genomic Loci in Human Cells
by an Optimized CRISPR/Cas system.Chen et al.
Cell 2013
• CRISPR interface for sequence specific control of
gene expression Larson et al. Nature protocols
2013
• Repurposing CRISPR as an RNA Guided Platform
for Sequence Specific Control of Gene Expression
et al Cell 2013