2. CRISPR Cas System
It is a genome editing tool that is creating a buzz in the
science world.
It acts as adaptive immune systems in bacteria & archaea.
It provides sequence-specific protection against foreign
invading elements ( viruses, phages & plasmids ) with both
DNA & RNA genomes.
CRISPR-Cas systems are highly diverse.
I. CRISPR ( Clustered Regularly Interspaced Short
Palindromic Repeats ) loci.
II. Cas ( CRISPR-associated ) proteins can target & cleave
invading DNA in a sequence-specific manner.
3. A CRISPR array is composed of a series of repeats
interspaced by spacer sequences acquired from invading
genomes.
The spacer sequences ( protospacers ) are variable &
originate from invading DNA.
CRISPR-Cas systems are found in the genomes of 40-50%
of bacteria.
4. Stages of CRISPR-Cas System
CRISPR-Cas immunity can be broken down into three stages:-
adaptation, expression & interference.
a) Adaptation :- Cas1 & Cas2 proteins are required for the
acquisition of DNA spacers by the CRISPR locus, & they display
polarity towards the leader sequence end of the array.
b) Expression :- The CRISPR array provides a precursor transcript
( precursor crRNA ) that is processed into mature crRNA
( CRISPR-RNA ) leading to the formation of crRNA-Cas
effector complexes.
c) Interference :- These complexes recognize & bind to the
complementary nucleic acids, resulting in the degradation of
the target molecule.
5.
6.
7. Classes of CRISPR-Cas Systems
These immunogenic systems are classified into two broad
classes on the basis of the crRNA-effector complexes.
I. Class 1 CRISPR-Cas systems have multi-subunit effector
complexes & are of types I, III, IV.
II. Class 2 CRISPR-Cas systems have a single protein & are of
types II, V, VI.
The 6 types can be broken down into more than 20
subtypes on the basis of gene content & locus
architecture.
8.
9. A particular feature of the associated multi-subunit effector
complexes of type III systems is the targeting of both ssRNA &
transcriptionally active DNA.
The effector complexes of type-IIIA & type-IIIB systems ( Csm &
Cmr complexes respectively ) have been found to have a common
mechanism of RNA-dependent DNA degradation.
Cas1 integrase is the key enzyme of the CRISPR-Cas adaptation
module that mediates acquisition of spacers derived from foreign
DNA by CRISPR arrays.
In diverse bacteria, the Cas1 gene is fused to a gene encoding a
reverse transcriptase (RT) related to group-II intron RTs.
An RT-Cas1 fusion protein has enable acquisition of CRISPR
spacers from RNA ( genomic RNA, plasmid RNA, DNA phage
transcript or RNA phage sequences ).
10. While the majority of CRISPR-Cas immune systems adapt
to foreign genetic elements by capturing segments of
invasive DNA, some systems carry reverse transcriptases
that enable adaptation to RNA molecules.
11.
12. CRISPR-Cas9 System
Type II CRISPR-Cas9 systems have been used in a variety of
organisms including microbes, fungi, plants & animals.
CRISPR-Cas9 system is a unique technology that enables
geneticists & medical researchers to edit parts of the
genome by removing, adding or altering sections of the
DNA sequence.
In the type II CRISPR-Cas9 systems, a Cas9 endonuclease &
a guide RNA establish a functional guide RNA-Cas9
complex.
The guide RNA consists of a DNA-targeting CRISPR-
associated RNA (crRNA) & the trans-activating crRNA
(tracrRNA).
13. Each crRNA hybridizes with a trans-activating crRNA
(tracrRNA) to form a single guide RNA (sgRNA).
14. The sgRNA then combines with the Cas9 nuclease &
directs Cas9 to cleave complementary target DNA
sequences adjacent to a protospacer-adjacent motif (PAM)
thereby creating a double-strand break in the DNA
sequence.
15. The CRISPR-Cas9 complex is recruited to the target DNA
site by its guide RNA ( which has a ~20 nucleotide
sequence complementary to its target ).
The endonuclease activity of Cas9 causes a double –strand
break at the target site.
Through the generation of a sequence-specific double-
strand break by Cas9 in the host, the error-prone DNA
repair pathway ( non-homologous end joining ) will be
triggered which often results in insertion/deletion of
mutations at the site of editing.
16.
17. The Cas9 nuclease is derived from Streptococcus pyogenes
& contains two active sites :-
1) The resistance to ultraviolet C (RuvC) endonuclease site
at the amino-terminal end.
2) The HNH (histidine-asparagine-histidine) endonuclease
site in the middle of the protein.
Both of the domains can cleave exogenous double-
stranded DNA.
The HNH nuclease domain cleaves the DNA strand that is
complementary to the crRNA.
The RuvC nuclease domain cleaves the DNA strand
opposite to the complementary strand.
