2. What is CRISPER/Cas9?
• CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic
Repeats.
• CRISPR is a family of DNA sequences in bacteria and archaea.
• These sequences play a key role in a prokaryotic defense system, and form the
basis of a technology known as CRISPR/Cas9 that effectively and specifically
changes genes within organisms.
• The CRISPR/Cas system is a prokaryotic immune system that confers resistance to
foreign genetic elements such as those present within plasmids and phages that
provides a form of acquired immunity.
• CRISPRs are found in approximately 50% of sequenced bacterial genomes and
nearly 90% of sequenced archaea.
• The Cas9 endonuclease is a four-component system that includes two small RNA
molecules named CRISPR RNA (crRNA) and trans-activating CRISPR RNA
(tracrRNA).
• CRISPR-Cas is the only adaptive immune system in prokaryotes known so far.
3. History of CRISPER/Cas9:
• 1987 - CRISPR repeats first observed in bacterial genoms.
• 2000 - CRISPR sequence are found to be common in other microbes.
• 2002 - Coined CRISPR name, defined signature Cas genes.
• 2006 - CRISPR proposed to be a bacterial adaptive immune system.
• 2007 - First experimental evidence for CRISPR adaptive immunity.
• 2010 - CRISPR/Cas9 developed as a gene editing tool.
• 2013 - First demonstration of Cas9 genome engineering in eukaryotic
cell.
• 2014 - Monkeys with CRISPR-engineered targeted mutations are
born.
• 2015 - CRISPR/Cas9 used to edit human embryos.
• 2017 - NAS reports outline criteria to be met for germline editing
clinical trials.
4. Classification of CRISPR/Cas:
• CRISPR-Cas systems can be divided into two main classes, which are
further classified into six types and several sub-types.
• The classification is based on the occurrence of effector Cas proteins
that convey immunity by cleaving foreign nucleic acids.
• In class 1 CRISPR-Cas systems (types I, III and IV), the effector module
consists of a multi-protein complex.
• In class 2 systems (types II, V and VI) use only one effector protein.
5. Use for gene editing
• A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been
modified to edit genomes.
• By delivering the Cas9 nuclease complexed with a synthetic guide
RNA (gRNA) into a cell, the cell's genome can be cut at a desired
location, allowing existing genes to be removed and/or new ones
added.
• The Cas9-gRNA complex corresponds with the CAS III CRISPR-RNA
complex in the above diagram.
6. Characteristics of genetic variation by
CRISPR
• Substitution and Deletion and insertion
• Loss of function and gain of function
• Transgenic process but non transgenic traits
7. 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 or maturation, and
(iii) target interference
8. Adaptation or spacer acquisition
• In first phase, a distinct sequence called a protospacer is
incorporated into the CRISPR array yielding a new spacer.
• Two proteins, Cas1 and Cas2, seem to be ubiquitously involved in the
spacer acquisition process as they can be found in almost all CRISPR-
Cas types.
• The CRISPR-Cas systems are composed of a cas operon and a CRISPR
array that comprises identical repeat sequences that are interspersed
by phage-derived spacers. Upon phage infection, a sequence of the
invading DNA (protospacer) is incorporated into the CRISPR array by
the Cas1–Cas2 complex.
• However, the mechanism of spacer acquisition is still not fully
understood.
Figure in slide number 11
9. crRNA biogenesis or maturation
• The CRISPR array is then transcribed into a long precursor CRISPR
RNA (pre-crRNA)..
• Pre-crRNA is further processed by Cas6 in type I and III systems
(processing in type I-C CRISPR-Cas systems by Cas5d).
• In type II CRISPR-Cas systems, crRNA maturation requires tracrRNA,
RNase III and Cas9.
• In type V-A systems Cpf1 alone is sufficient for crRNA maturation.
Figure in slide number 11
10. Target interference
• In the interference state of type I systems, Cascade is guided by
crRNA to bind the foreign DNA in a sequence-specific manner and
subsequently recruits Cas3 that degrades the displaced strand
through its 3′–5′ exonucleolytic activity.
• Type III-A and type III-B CRISPR-Cas systems employ Csm and Cmr
complexes, respectively, for cleavage of DNA (red triangles) and its
transcripts (black triangles).
• A ribonucleoprotein complex consisting of Cas9 and a tracrRNA :
crRNA duplex targets and cleaves invading DNA in type II CRISPR-Cas
systems.
• The crRNA-guided effector protein Cpf1 is responsible for target
degradation in type V systems.
Figure in slide number 11
11. Fig:Simplified model of the immunity mechanisms of class 1 and class 2 CRISPR-Cas systems.
# cas operon (blue arrows)
#Identical repeat sequences
(black rectangles)
#Phage-derived spacers
(coloured rectangles)
12. Graphical representation of the
CRISPR-Cas9 system
Step 1. Adaptation – DNA from the invading
virus is processed into short segments. These
segments are inserted into the CRISPR
sequence to function as new spacers.
Step 2. Production of CRISPR RNA – the DNA
undergoes a transcription process that copies
DNA into RNA. The single-stranded RNA is cut
into short pieces called CRISPR RNAs.
Step 3. Targeting – CRISPR RNAs are
programmed to destroy the viral material.
Here, the ‘RNA sequences’ are copied from the
viral DNA sequences.
13. Advantages
• Gene specific mutation
• Efficient production of desirable mutation
• 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.
• 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.
14. Disadvantages
• Necessity of knowing gene function and sequences.
• Prerequisite of efficient genetic transformation.
• Ethical concerns.
• Since the scope of the DNA repair system is not to integrate DNA fragments in
the genome, targeted alleles often carry additional modifications, such as
deletions, partial or multiple integrations of the targeting vector, and even
duplications.
• Secondary unwanted mutational events .
• Target sequences may be limited due to PAM sequences.
• Off target sequences could be possible.
15. Application
• CRISPR/Cas9 in cancer drug development.
• CRISPR/Cas9 mediated chromatin immunoprecipitation.
• Epigenetic editing with CRISPR/Cas9.
• Live imaging of DNA or mRNA with CRISPR/Cas9.
• CRISPR technologies for transcriptional activation and repression.
• CRISPR/Cas9 in cancer modeling.
• CRISPR/Cas9 therapeutic applications.
• CRISPR/Cas9 in different types of cancer treatment.
• Allow scientists to adequately edit genes to cure diseases for plant
and animal species as well as human.
16. CRISPR/Cas9 in Bangladesh
• Some scientists have actively started thinking and acting on using CRISPR-Cas9
system application in their research work. This includes initiatives being taken by
the Plant Biotechnology Laboratory at the Department of Biochemistry and
Molecular Biology at the University of Dhaka.
• An international network of scientists, dedicated to the cause of redressing blast
attacks in wheat in South America and Bangladesh, are also toying with the idea
of applying genome editing so that blast-causing fungi can no longer attack
wheat fields.
• With a goal to expose our scientists to a new world of enormous possibilities,
the University Grants Commission (UGC), Krishi Gobeshona Foundation (KGF),
University of Dhaka (DU), Faculty of Biological Sciences, DU, National Institute of
Biotechnology (NIB) and Bangladesh Council of Scientific and Industrial Research
(BCSIR) helped Global Network of Bangladeshi Biotechnologists (GNOBB) to
organise a workshop on CRISPR-Cas9 technology and its application in genome
editing on 2017. This workshop, organised by GNOBB, is giving the country's
scientists a much needed exposure to the highly potential new science. Experts
from Japan will be leading this workshop for bench scientists from the
agricultural research institutes as well as NIB, BCSIR and public and private
universities.
