Crispr: Presented By: Anurag Chauhan (21513) M.Sc. Microbial-Biotechnology - IV Department of Biotechnology

CRISPR Cas-9
Presented by : Anurag Chauhan (21513)

M.Sc. Microbial-Biotechnology - IV
Department of Biotechnology
CRISPRs
Clustered Regularly Interspaced Short Palindromic Repeats
 CRISPRs are DNA loci containing short repetitions of base sequences
which separated by short "spacer DNA" from previous exposures to
a virus or phage. CRISPR is a family of DNA sequences found within
the genomes of prokaryotic organisms such as bacteria and
archaea. And these sequences play a key role in the antiviral
defence system of prokaryotes.
 Cas9 (CRISPR associated protein 9) is a protein which plays a
vital role in the immunological defence of certain bacteria against
DNA viruses, and which is heavily utilized in genetic engineering
applications. Its main function is to cut DNA and therefore it can
alter a cell's genome. Cas9 is an RNA-guided DNA endonuclease
enzyme associated with the CRISPR.
 CRISPR/Cas9 technique is a type of genetic engineering in which
DNA is inserted, replaced, or removed from a genome using Cas9
endonucleases (molecular scissors).
HISTORY OF CRISPR
Components of CRISPR cas9
 Protospacer- sequence present in virus or plasmid, adjacent to PAM,
recognized by cas proteins as their target.
 Spacer- Novel sequences of protospacer, acquired by bacteria and present
between repeats.
 PAM-Protospacer adjacent motif(PAM) is a 2-6 bp DNA sequence immediately
following the DNA sequence targeted by cas nucleases. PAM is a component
of the invading virus or plasmid, but is not a component of the bacterial
CRISPR locus.
 crRNA-It contains by the guide RNA that locates the correct section of host
DNA along with a region that binds to tracrRNA.
 Trans activating crRNA (tracr RNA)- It is a small trans-encoded RNA found in
type-ii CRISPR system. It is binds to crRNA and forms an active complex.
 Cas9- It is a RNA directed DNA endonuclease.
 sgRNA-Single guide RNAs are a combined RNA consisting of a tracrRNA and at
least one crRNA.
Classification of CRISPR cas
 The class1 systems use a large complex of Cas proteins for crRNA guided
targeting. (Andersson and Banfield, 2008)
 The class2 system requires only a single protein for RNA-guided DNA
recognition and cleavage. (M. Jinek et al.,2012)
Structure of Cas9 Protein
Structure of single guide RNA (sgRNA)
Working Mechanism
 The CRISPR-Cas9 system consists of two key molecules that introduce a
change into the DNA. These are:
Cas9 : This consists of a small piece of pre-designed RNA sequence (about 20
bases long) located within a longer RNA scaffold. The scaffold part binds to
DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the
genome. This makes sure that the Cas9 enzyme cuts at the right point in the
genome.
Guide RNA (gRNA) : It acts as a pair of ‘molecular scissors’ that can cut the
two strands of DNA at a specific location in the genome so that bits of DNA
can then be added or removed.
The guide RNA is designed to find and bind to a specific sequence in the DNA.
The guide RNA has RNA bases that are complementary to those of the target
DNA sequence in the genome. Hence, the guide RNA will only bind to the
target sequence and no other regions of the genome.
The Cas9 follows the guide RNA to the
same location in the DNA sequence
and makes a cut across both strands of
the DNA.
At this stage the cell recognises that the

DNA is damaged and tries to repair it.
Scientists can use the DNA repair

machinery to introduce changes to one
or more genes in the genome of a cell
of interest.
ADVANTAGES
 Target design simplicity- Because the target specificity relies on ribonucleotide
complex formation and not protein/DNA recognition, gRNAs can be designed
readily and cheaply to target nearly any sequence in the genome specifically.
 Efficiency- The system is super-efficient. Modifications can be introduced by
directly injecting RNAs encoding the Cas protein and gRNA into developing
mouse embryos. This eliminates the long and laborious processes of transfecting
and selecting mouse ES cells that are required to create targeted mutant mice
using classical homologous recombination techniques.
 Multiplexed mutations- Mutations can be introduced in multiple genes at the
same time by injecting them with multiple gRNAs. JAX Assistant Professor Dr.
Haoyi Wang and his former colleagues in Rudolf Jaenisch’s group at the
Whitehead Institute recently reported using the CRISPR/Cas system to
successfully introducing mutations in five different genes in mouse ES cells
simultaneously.
DISADVANTAGES
 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.
 When performing the CRISPR/Cas9 procedure directly on embryos,
on the other hand, it is impossible to select for the desired event,
greatly limiting the possibility to identify the desired allele.
 One drawback to the CRISPR/Cas9 system in plants concerns off-
target effects.
 Risky human experimentation. Altering one gene could have
unforeseen and wide spread effects on other parts of the genome .
APPLICATIONS
 CANCER IMMUNOTHERAPY : The screening for 2368 genes via the CRISPR–Cas9 system in
melanoma cells revealed a new targeted therapy for immunotherapy of cancer.
 INHIBITING RETINAL ANGIOGENESIS : Using the CRISPR–Cas9 system and the adeno‐associated
virus (AAV) viral vector, researchers were able to prevent the angiogenesis in the clinical model
for the first time, resulting in loss of vision.
 HEART FAILURE : A study was conducted to investigate the correction of a pathogenic gene
mutation (MYBPC3) in a human embryo with hypertrophic cardiomyopathy (a disease that
ultimately causes heart failure).
 Programmable Nucleases as Tools for Efficient and Precise Genome Editing : CRISPR is very
precise tool for the specific cleavage the target sequence of DNA so use in the various genetic
disorders like sickle cell anemia .
 Completely curing bacterial blight in rice which is caused by Xanthomonas oryzae.
 CRISPR “could be used to modify disease-causing genes in embryos brought to term, removing
the faulty script from the genetic code of that person’s future descendants as well.
 FUTURE Applications : Remove malaria from mosquitos, Treating Alzheimer’s disease, Treating
HIV, Develop new drugs, Livestock (CRISPR/Cas9 has been utilized in China to delete genes),
Agricultural crops- to improve crop disease resistance and environmental stress tolerance in
plants, Develop new cancer treatments -modify immune cells, Reduce our need for plastic-
CRISPR can be used to manipulate a type of yeast that transforms sugars into hydrocarbons,
which can be used to make plastic .
CONCLUSION
CRISPR/Cas9 genome engineering technology has provided researchers with

an invaluable tool to accelerate the generation of mouse models for
biomedical in vivo research. The furious pace of CRISPR development,
combined to its versatility and ease of use, have already left a mark in the field
of molecular genetics. Its combination with established technologies will
greatly expand opportunities for the generation of new and valuable
genetically engineered mouse models for basic and translational research.
THANK YOU…

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CRISPR Cas-9

Presented by : Anurag Chauhan (21513)

At this stage the cell recognises that the

Scientists can use the DNA repair

CRISPR/Cas9 genome engineering technology has provided researchers with

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