Animal Biotechnology

Theory Assignment
Topic
“ CRISPR-Cas9 ”

By
Ishika Tyagi
B.Sc. Life Science (Sem-5)
Roll No. – 19/12064
 Group-3
Contents
● Introduction
● History
● CRISPR/Cas Systems
● Overview of the CRISPR technique of genome editing
● General Mechanism
● Types of CRISPR/Cas Systems
● Applications of CRISPR/Cas9
● Ethical Challenges
● Conclusion
● Refrences
 Introduction

Human genome editing is a powerful tool which offers great scientific and
therapeutic potential.
The CRISP-Cas9 system is a powerful genome editing technology with the
potential to create a variety of novel therapeutics for a range of diseases, many
of which are currently untreatable.
CRISPR/Cas systems are highly diverse adaptive microbial immune systems
used by most archaea (90%) and many eubacteria (40%) to protect themselves
from invading viruses & plasmids.

History

● 1987 - first report of CRISPR clustered repeats

● 2000 - recognition that CRISPR families are present throughout
prokaryotes
● 2002 - coined “CRISPR” name, defined signature Cas genes
● 2005 - identified foreign origin of spacers, proposed adaptive immunity
function
● 2007 - first experimental evidence for CRISPR adaptive immunity
● 2008 - CRISPR acts upon DNA targets
● 2009 - type III-B Cmr CRISPR complexes cleave RNA
● 2010 - Cas9 guided by spacer sequences and cleaves target DNA via
DSBs
● 2011 - tracrRNA forms a duplex structure with crRNA in association
with Cas9
type II CRISPR systems are modular and can be heterologously
expressed in other organisms
● 2012 - in vitro characterization of DNA targeting by Cas9
● 2013 - first demonstration of Cas9 genome engineering in eukaryotic
cells
● 2014 - genome-wide functional screening with Cas9
crystal structure of apo-Cas9
 crystal structure of Cas9 in complex with guide RNA and target DNA

CRISPR/Cas systems

CRISPRS (Clustered Regularly Interspaced Short Palindromic Repeats) consist

of highly conserved short repeated sequences separated by similarly sized short
spacers sequences. The size of CRISPR repeats and spacers varies between 23
to 47 bp and 21 to 72 bp, respectively.
The bacterial genome may contain more than one CRISPR locus. The CRISPR
loci have highly diverse and hypervariable spacer sequences, even between
closely related strains. Spacers are unique sequences originating from viral or
plasmid DNA. These sequences are used as recognition elements and by adding
new spacers new matching viral or plasmid genomes are recognized and
destroyed.
Another feature associated with CRISPR loci is the presence of a conserved
sequence, called leader, located upstream of the CRISPR with respect to the
direction of transcription. CRISPR activity requires the presence of a set of
CRISPR-associated (cas) genes, usually found adjacent to the CRISPR, that
code for Cas proteins essential to the immune response. The cas genes encode
proteins with a variety of functions such as nucleases, helicases and
polymerases. The CRISPR immune system works through the cooperation of
many diverse Cas proteins.
 Overview of the CRISPR technique of genome
editing

Caso (blue) utilizes a guide RNA (purple) that can recognize and base-pair with
a target genomic sequence (thick red strands). Caso also requires a short
sequence, known as a PAM sequence (orange), downstream from the target
sequence. Upon finding a matching sequence, Cas9 cuts both strands of target
DNA, resulting in a double-stranded break that is repaired rapidly by DNA
repair enzymes. CRISPR can be used to edit, delete, or add new DNA to a
specific location in the genome.
 General Mechanism

A simple analogy to understand the CRISPR mechanism is to imagine the

design of a personalized weapon.
Suppose, Earth is attacked by an alien species and none of the earthly weapons
are working on it, so someone would go and pick up the body parts of alien and
based on that a new weapon specific for that alien would be developed. Also,
some of the body parts will be stored for future memory.
Similar to this, a bacterium, when attacked by bacteriophage, is not aware of
any defence mechanism. Hence, a small Cas protein picks the part of DNA of
phage and bring it close to the CRISPR loci, where it is integrated into the
CRISPR site.
A crRNA (CRISPR RNA) is synthesized from that loci which along with other
proteins then recognize the complementary sequence present in the invading
DNA of phage and degrade it. The CRISPR/Cas system works in a similar
manner.

The general mechanism of CRISPR works three distinct phases:

Adaptation (or acquisition) Phase: Adaptation is the first step in the process
of developing a weapon against foreign invading DNA (viral or plasmid DNA).
It involves picking up a piece of invading DNA (termed protospacers) and
integrating it between two adjacent repeats in the CRISPR locus. The invading
DNAs are directionally integrated, as new CRISPR spacers, into a CRISPR
array that is separated by repeat sequences, thus creating a memory of the
invading genetic elements.
Expression and maturation (or biogenesis) Phase: Once the desired sequence
(i.e, protospacer) from the invading DNA is integrated into the loci of CRISPR,
the CRISPR locus is transcribed into a pre-CRISPR RNA (pre-crRNA),
CRISPR loci are transcribed from an upstream promoter located in the AT-rich
leader sequence. The pre CrRNA is then processed into mature crRNAS, each
containing a transcribed spacer sequence joined to the partial repeat sequence.
Interference (or targeting) Phase: The crRNA forms a complex with Cas
proteins. The crRNA of the crRNA-Cas complex makes base pairing with the
protospacer of the invading DNA. Finally, crRNA-directed cleavage of
invading DNA occurs by Cas proteins at protospacer, a site complementary to
the crRNA spacer sequence.
 Types of CRISPR/Cas Systems

There are three major types of CRISPR/Cas system- type I, type II and type III
which have been identified and these can be further divided into several
subtypes that encompass considerable structural and functional diversity.
● Type I systems are the most frequent and widespread systems, which
target invading DNA in a Cascade (CRISPR-associated complex for
antiviral defence) complex driven and Protospacer Adjacent Motif
(PAM)-dependent manner.
● The PAM is a short sequence motif located adjacent to the protospacer on
the invading DNA. It is essential for recognition, cleavage and the
distinction between self and non-self DNA.
● The absence of the PAM sequence at the CRISPR locus in the host
genome protects it from self-cleavage.
● In type 1 system, pre-crRNA cleavage is carried out by Cas
endoribonuclease, Cas6.
● Mature crRNA makes ribonucleoprotein complex with Cascade. During
the interference stage, the cleavage of invading target DNA is carried out
by recruiting a Cas nuclease, Cas3.

● Type II systems work in a tracrRNA (a small trans noncoding RNA) and

PAM dependent manner.
● In this system, pre-crRNA processing involves the expression of a
tracrRNA, which base-pairs with the repeats in the pre-crRNA transcript.
● The resulting duplexes are cleaved in the repeat sequences by RNase III
in a Cas9-dependent reaction.
● The tracrRNA remains bound to the crRNA and the mature crRNA-
tracrRNA duplexes are complexed with Cas9.
● The cleavage of invading target DNA is carried out by the Cas-crRNA
ribonucleoprotein complex itself.
● Type III systems work in PAM-independent manner.
● Pre-crRNA cleavage is carried out by Cas endoribonuclease, Cas6.
Mature crRNA makes ribonucleoprotein complex with Csm in type III-A
systems and Cmr in type III-B systems.
● The cleavage of invading target DNA is carried out either by the Cas-
crRNA ribonucleoprotein complex itself (in type III-B system) or by
recruiting a Cas nuclease, probably Cas6 (in type III-A systems).
● The two types of type III systems target either dsDNA (type III-A
systems) or RNA (type III-B systems).
 Applications of CRISPR Cas9

Antibiotic Resistance
CRISPR-Cas9 has been shown to effectively target and eliminate bacterial
species, including antibiotic resistant strains, from a community of bacteria.
This precise targeting allows the elimination of harmful bacteria, but avoids
beneficial bacteria.

Diabetes
The California Institute for Regenerative Medicine (CIRM) awarded a grant to
researchers at Children’s Hospital Los Angeles who are using CRISPR-Cas9 to
develop a personalized approach for treating genetic forms of diabetes. 
 Ethical Challenges

Recently, the mutagenic chain reaction (MCR) based on the CRISPR/Cas9

system, has provided a novel and prospective way to generate homozygous loss
of function mutations by autocatalysis, as confirmed in Drosophila.
Recently, a paper reporting gene editing in human embryos was published in the
journal Protein & Cell, which raised concerns about the ethics of employing the
CRISPR/Cas9 system. Thereafter, both the editorial team of Nature and Science
announced that although the CRISPR/Cas9 system shows huge potential for
genome editing, its use for modifying human germline cells should be
considered very seriously, and progressive policy on this issue should be
developed.

Conclusion

The rapidly developing suite of CRISPR-Cas9-mediated gene-editing

technologies is yielding enormously beneficial results in the modern life
sciences. These tools allow for precise genomic modifications, transcriptional
regulation, and epigenetic editing with high efficiency, generality, and
simplicity. Undoubtedly, CRISPR systems are endowed with the potential to
revolutionize the fields of genetics and medicine.
The past few years have witnessed the introduction of metadata era with large-
scale analyses of genome sequences,94,95 deep sequencing technologies,96,97
and single-cell transcriptomics.98 CRISPR-based technologies are suggested to
play a major role in this metadata revolution. The technologies will help in
understanding specific gene functions through their ability to precisely dissect
genetic networks.

References

● Pranav Kumar Molecular biology Fundamentals & techniques

● www.genescript.com
● www.slideshare.net
● www.cell.com
● pubmed.ncbi.nlm.nih.gov