Crispr/Cas 9 A New Tool For Genome Editing

1. CRISPR/CAS 9
A NEW TOOL FOR GENOME EDITING…
Dayalbagh
Educational Institute, Agra
PRESENTED BY
SHIVANI SINGH
M.SC FINAL SEM

2. CONTENT
 INTRODUCTION
 PIONEER SCIENISTS
 HISTORY
 BACKGROUND
 DEFINATION
 GENERAL IDEA BEHIND CRISPR
 MECHANISM OF CRISPR/ CAS
 USES OF CRISPR/CAS 9

3. INTRODUCTION
CRISPER stands for
Clustered regularly interspaced short
palindromic repeats.
What is Cas 9?
Cas9 (CRISPR associated protein 9, formerly called Cas5,
Csn1, or Csx12) is a 160 kD protein which plays a vital role
in the immunological defense of certain bacteria against
DNA viruses and plasmids.

4. 16 CRISPR Scientists & Pioneers: The Real Heroes
of Genome Engineering
 Jennifer Doudna: The Mother of CRISPR
 Emmanuelle Charpentier: CRISPR Co-Inventor
 Feng Zhang: The Man Who Dared to Move Away from Bacteria
 George Church: The Father of Genome Engineering
 Francisco Mojica: Discovered the Existence of CRISPR Sequence in
Bacteria
 Matthew Porteus: The Pioneer of Cell-Based CRISPR Therapies
 David Liu: Introducing Base-Editor Enzymes
 Stephen Tsang: Pioneer of Ophthalmological Genome Surgery Options

5. continued........
 Kevin Esvelt: Changing the Game with Responsible Gene Drives
 Prashant Mali: Taking the Multidisciplinary Approach
 Stanley Qi: Developing the Next Stage of CRISPR Tech
 Patrick Hsu: Expanding CRISPR Toolkit to RNA
 Michel Sadelain: Novel Therapies Targeting Cancer
 Andy Scharenberg: Editing T Cells
 Jacob Corn: The Intersection of Genome Editing and DNA Repair
 Alison Van Eenennaam: Using CRISPR to Edit Cattle

6. HISTORY
Where do CRISPR come from?
 CRISPR was first discovered in archaea (and later in bacteria) by Francisco Mojica, a
scientist at the University of Alicante in Spain. He proposed that CRISPRs serve as part of
the bacterial immune system, defending against invading viruses. They consist of
repeating sequences of genetic code, interrupted by “spacer” sequences – remnants of
genetic code from past invaders. The system serves as a genetic memory that helps the
cell detect and destroy invaders (called “bacteriophage”) when they return. Mojica’s
theory was experimentally demonstrated in 2007 by a team of scientists led by Philippe
Horvath.
 In January 2013, the Zhang lab published the first method to engineer CRISPR to edit
the genome in mouse and human cells.

7. BACKGROUND
 How was it developed?
• Some bacteria have a similar, built-in, gene editing system to the
CRISPR-Cas9 system that they use to respond to invading
pathogens like viruses, much like an immune system.
• Using CRISPR the bacteria snip out parts of the virus DNA and
keep a bit of it behind to help them recognize and defend against
the virus next time it attacks.
• Scientists adapted this system so that it could be used in other
cells from animals, including mice and humans.

8. CRISPR-Cas9
Definition
CRISPR/Cas9 is a technique that
allows for the highly specific and
rapid modification of DNA in a
genome, the complete set of genetic
instructions in an organism.

9. GENERAL IDEA BEHIND CRISPR
 CRISPR-Cas immunity is a natural process of bacteria and archaea.
CRISPR-Cas prevents bacteriophage infection, conjugation and
natural transformation by degrading foreign nucleic acids that
enter the cell.
 CRISPR/Cas9 gene targeting requires a custom single guide RNA
(sgRNA) that contains a targeting sequence (crRNA sequence) and
a Cas9 nuclease-recruiting sequence (tracrRNA).

10.  Specialized Cas proteins snip foreign DNA into small fragments
approximately 20 bp in length and paste them into contiguous stretches of
DNA known as CRISPR arrays. Separate Cas proteins then express and
process the CRISPR loci to generate CRISPR RNAs (crRNAs). Through
sequence homology, these crRNAs guide a Cas nuclease to the specified
exogenous genetic material, which must also contain a species-specific
sequence known as a protospacer adjacent motif (PAM). The CRISPR
complex binds to the foreign DNA and cleaves it to destroy the invader .
CONTINUED.....

11. Mechanism of CRISPR

12. CONTINUED.. BASIC TERMS RELATED TO
CRISPR CAS SYSTEM
 are used in prokaryotic DNA editing involving CRISPR and
Cas9. These gRNAs are non coding short RNA sequences which bind to the complementary
target DNA sequences. Guide RNA first binds to the Cas9 enzyme and the gRNA sequence
guides the complex via pairing to a specific location on the DNA, where Cas9 performs its
endonuclease activity by cutting the target DNA strand.
 is a region of non-coding DNA between genes. The terms intergenic spacer
(IGS) or non-transcribed spacer (NGS) are used particularly for the spacer DNA between the
many tandemly repeated copies of the ribosomal RNA genes. In bacteria, spacer DNA sequences
are only a few nucleotides long.
 A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the
DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.

13. MOLECULAR MECHANISMS: ADAPTATION,
MATURATION AND INTERFERENCE
 The CRISPR-Cas system acts in a sequence-specific manner by
recognizing and cleaving foreign DNA or RNA. The defence
mechanism can be divided into three stages:
 (i) adaptation or spacer acquisition,
 (ii) crRNA biogenesis, and
 (iii) target interference

14. Acquisition of a new bacteriophage-derived spacer. Invading by a
bacteriophage for the first time, a new spacer derived from the
invading DNA and a repeat were incorporated into the CRISPR locus.

15. The immune mechanism of CRISPR-Cas system resisted invading DNA. When
the same bacteriophage invaded again, the CRISPR locus could transcribe
into a long pre-crRNA that was processed and generated small crRNAs. The
specific crRNA served as a guide for Cas proteins that recognized and
cleaved invading DNA.

16. Applications and Implications
 CRISPR-Cas9 has a lot of potential as a tool for treating a range of
medical conditions that have a genetic component, including
cancer, hepatitis B or even high cholesterol.
 Many of the proposed applications involve editing the genomes of
somatic (non-reproductive) cells but there has been a lot of
interest in and debate about the potential to edit germline
(reproductive) cells.

17. continued.....
 Because any changes made in germline cells will be passed on
from generation to generation it has important ethical
implications.
 The CRISPR/Cas9 is becoming a user-friendly tool for development
of non-transgenic genome edited crop plants to counteract
harmful effects from climate change and ensure future food
security of increasing population in tropical countries.
 Potentials of CRISPR/Cas9 for improvement of crops cultivated in
tropical climates to gain resiliency against emerging pests and
abiotic stresses.

18. Applications of CRISPR CAS in
agriculture field...
Applications of
CRISPR/Cas9: CRISPR/Cas9
is currently used in genetic
engineering, agriculture,
and nutritional research.

19. Better targeting of CRISPR-Cas9
 In most cases the guide RNA consists of a specific sequence of 20 bases.
These are complementary to the target sequence in the gene to be edited.
However, not all 20 bases need to match for the guide RNA to be able to
bind.
 The problem with this is that a sequence with, for example, 19 of the 20
complementary bases may exist somewhere completely different in the
genome. This means there is potential for the guide RNA to bind there
instead of or as well as at the target sequence.
 The Cas9 enzyme will then cut at the wrong site and end up introducing
a mutation in the wrong location. While this mutation may not matter at
all to the individual, it could affect a crucial gene or another important
part of the genome.

20. CONTINUE.....
 Scientists are keen to find a way to ensure that the CRISPR-Cas9 binds
and cuts accurately. Two ways this may be achieved are through:
 The design of better, more specific guide RNAs using our knowledge of
the DNA sequence of the genome and the 'off-target' behaviour of
different versions of the Cas9-gRNA complex.
 The use of a Cas9 enzyme that will only cut a single strand of the target
DNA rather than the double strand. This means that two Cas9 enzymes
and two guide RNAs have to be in the same place for the cut to be
made. This reduces the probability of the cut being made in the wrong
place.

21. Different types of crispr cas system
1. CRISPR cas 9
 Organisms: Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, Neisseria
meningitidis, Campylobacter jejuni
 Size: ~1,000–1,600 aa
 Guide spacer length: 18–24 nt
 Total guide length: ~100 nt (sgRNA)
 Cut: Blunt-ended dsDNA break
 PAM: 3-NGG (SpCas9), 3-NNGRRT (SaCas9), 3-NNNNGATT (NmCas9)
 The first and best-characterized single-protein CRISPR effector. Cas9 makes a blunt double-stranded
DNA break, which can then be repaired by either non-homologous end joining or homologous
recombination with a donor template DNA to create site-specific edits. Type II-A Cas9s generally have
high genome editing efficiency, but off-target cleavage at unintended genome sites can be a
disadvantage. Variants have been engineered to overcome these limitations, and type II-C Cas9s tend
to have naturally higher fidelity.

22. 2. CRISPR-Cas12
 Organisms: Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., Prevotella sp.
 Size: ~1,100–1,300 aa
 Guide spacer length: 18–25 nt
 Total guide length: 42–44 nt
 PAM: 5-TTTN (FnCas12a)
 Cut: 5 nt 5 overhang dsDNA break
 Cas12 is a compact and efficient enzyme that creates staggered cuts in dsDNA. Cas12
processes its own guide RNAs, leading to increased multiplexing ability. Cas12 has also been
engineered as a platform for epigenome editing, and it was recently discovered that Cas12a
can indiscriminately chop up single-stranded DNA once activated by a target DNA molecule
matching its spacer sequence. This property makes Cas12a a powerful tool for detecting tiny
amounts of target DNA in a mixture.

23. 3. CRISPR-Cas13
Also known as C2c2 or CasRx
 Organisms: Leptotrichia buccalis, Leptotrichia shahii, Ruminococcus flavefaciens, Bergeyella
zoohelcum, Prevotella buccae, Listeria seeligeri
 Size: ~900—1,300 aa
 Guide spacer length: 22–30 nt
 Total guide length: 52–66 nt
 Cut: ssRNA
 PAM: 3-H (LshCas13a), 5-D and 3-NAN or NNA (BzCas13b), none (RfCas13d)
 Cas13 is an outlier in the CRISPR world because it targets RNA, not DNA. Once it is activated by a
ssRNA sequence bearing complementarity to its crRNA spacer, it unleashes a nonspecific RNase
activity and destroys all nearby RNA regardless of their sequence. This property has been
harnessed in vitro for precision diagnostics. These systems can also be used for efficient,
multiplexable, and specific RNA knockdown or RNA sequence editing in mammalian cells. This
makes Cas13 a potentially significant therapeutic for influencing gene expression without altering
genome sequence.

24. The future of CRISPR-Cas9
 Much research is still focusing on its use in animal models or isolated human
cells, with the aim to eventually use the technology to routinely treat diseases
in humans.
 There is a lot of work focusing on eliminating ‘off-target’ effects, where the
CRISPR-Cas9 system cuts at a different gene to the one that was intended to be
edited.
 The rapid progress in developing Cas9 into a set of tools for cell and molecular
biology research has been remarkable, likely due to the simplicity, high
efficiency and versatility of the system. Of the designer nuclease systems
currently available for precision genome engineering, the CRISPR/Cas system is
by far the most user friendly. It is now also clear that Cas9’s potential reaches
beyond DNA cleavage, and its usefulness for genome locus-specific recruitment
of proteins will likely only be limited by our imagination.

25. References
 www.yourgenome.org
 royalsocietypublishing.org
 www.researchgate.net
 google images
 www.sciencedirect.com
 wikipedia
 www.synthego.com
 www.cell.com

26. Thank you .....