CRISPR-Cas9: A Potential Tool for Genome Editing 1. Introduction 1.1. Origins In the year 1987, a team of Japanese scientists were the first to describe an unusual locus found in the E. coli genome, adjacent to the iap gene, having short palindromic repeats interspersed by similarly sized non-repetitive DNA spacers (Fig. 1) [1]. This clustered, regularly interspersed, short palindromic repeat locus is therefore termed “CRISPR”. Nearly a decade later, up to forty percent of sequenced bacteria and ninety percent of archaea were found to harbor this CRISPR locus [2] [3]. In the year 2002, bacterial strains that survived bacteriophage infection were observed to express the CRISPR loci, suggesting that the particular region may have a role in prokaryotic adaptive immunity [4]. This hypothesis was subsequently confirmed when phage-resistant bacterial strains had specific CRISPR loci spacers disrupted, they acquired susceptibility to phage infection, while insertion of novel spacers into wild type stains demonstrated acquired resistance [5]. The proteins involved in the prokaryotic immune system were found to be conserved and encoded in close proximity to the CRISPR locus (Fig. 1) [6]. As such, these proteins are known as CRISPR-associated, or in short, “Cas”. 1.2. Mechanism of Action The bacterial CRISPR-based acquired immunity (Fig. 1) encompasses three processes, namely, spacer addition, CRISPR-RNA (crRNA) maturation and target elimination [7][8][9]. Upon initial exposure to foreign phage DNA, bacterial Cas1 locates the DNA’s unique protospacer adjacent motif (PAM) and cuts a short DNA fragment (protospacer) that is directly beside [7]. Integration of this protospacer into the CIRSPR locus is directed by the Cas1-Cas2 complex [7]. Successful integration of protospacers into the host genome are thereafter referred to as spacers. A pre-crRNA is a long mRNA transcript of the CRISPR locus containing an array of spacer and repeat sequences. It hybridizes with multiple trans-activating crRNAs (tracrRNA) to form RNA duplex structures that are targeted for cleavage by RNase III [10]. The cleaved, mature crRNA, encodes for a particular spacer and repeat sequence which remains hybridized to a tracrRNA, and this short duplex is called the guide RNA (gRNA) [7][8]. Each gRNA has a unique spacer sequence that recognizes its complementary protospacer of the phage DNA [7][8]. When an immunized bacterial cell reencounters the same phage DNA, the appropriate gRNA guides Cas9, an endonuclease, to specifically target and eliminate the invading DNA by inducing a site-specific double strand break (DSB) [7][8]. The PAM ensures that Cas9
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CRISPR-Cas9 Review: A potential tool for genome editing
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CRISPR-Cas9: A Potential Tool for Genome Editing
1. Introduction
1.1. Origins
In the year 1987, a team of Japanese scientists were the first to describe an unusual locus
found in the E. coli genome, adjacent to the iap gene, having short palindromic repeats interspersed
by similarly sized non-repetitive DNA spacers (Fig. 1) [1]. This clustered, regularly interspersed,
short palindromic repeat locus is therefore termed “CRISPR”. Nearly a decade later, up to forty
percent of sequenced bacteria and ninety percent of archaea were found to harbor this CRISPR locus
[2] [3].
In the year 2002, bacterial strains that survived bacteriophage infection were observed to
express the CRISPR loci, suggesting that the particular region may have a role in prokaryotic adaptive
immunity [4]. This hypothesis was subsequently confirmed when phage-resistant bacterial strains had
specific CRISPR loci spacers disrupted, they acquired susceptibility to phage infection, while
insertion of novel spacers into wild type stains demonstrated acquired resistance [5].
The proteins involved in the prokaryotic immune system were found to be conserved and
encoded in close proximity to the CRISPR locus (Fig. 1) [6]. As such, these proteins are known as
CRISPR-associated, or in short, “Cas”.
1.2. Mechanism of Action
The bacterial CRISPR-based acquired immunity (Fig. 1) encompasses three processes,
namely, spacer addition, CRISPR-RNA (crRNA) maturation and target elimination [7][8][9].
Upon initial exposure to foreign phage DNA, bacterial Cas1 locates the DNA’s unique
protospacer adjacent motif (PAM) and cuts a short DNA fragment (protospacer) that is directly beside
[7]. Integration of this protospacer into the CIRSPR locus is directed by the Cas1-Cas2 complex [7].
Successful integration of protospacers into the host genome are thereafter referred to as spacers.
A pre-crRNA is a long mRNA transcript of the CRISPR locus containing an array of spacer
and repeat sequences. It hybridizes with multiple trans-activating crRNAs (tracrRNA) to form RNA
duplex structures that are targeted for cleavage by RNase III [10]. The cleaved, mature crRNA,
encodes for a particular spacer and repeat sequence which remains hybridized to a tracrRNA, and this
short duplex is called the guide RNA (gRNA) [7][8].
Each gRNA has a unique spacer sequence that recognizes its complementary protospacer of
the phage DNA [7][8]. When an immunized bacterial cell reencounters the same phage DNA, the
appropriate gRNA guides Cas9, an endonuclease, to specifically target and eliminate the invading
DNA by inducing a site-specific double strand break (DSB) [7][8]. The PAM ensures that Cas9
complexes locate the correct protospacer sequence [7]. As the PAM is only found on invading phage
DNA, the DSB mechanism is able to discriminate self from non-self [7].
Figure 1: Bacterial CRISPR-based acquired immunity. Phase 1 (immunization): Initial injection of phage double strand DNA into a
bacterial cell followed by Cas1-Cas2 complex (large teal oval with associated grey ovals) excision of the protospacer (yellow rectangle)
from the phage DNA. The Cas1-Cas2 complex inserts the protospacer into the Type II CRISPR locus region containing short palindromic
repeats (black) and novel spacers derived from other foreign phage DNA (purple, green and red rectangles). Phase 2 (immunity): Pre-crRNA
(multi-colored linear mRNA) and tracrRNA (purple stem-loop mRNA) are transcribed and processed into guide RNA (mature crRNA-
tracrRNA duplex) which subsequently guides Cas9 (small teal oval) to cleave the invading DNA. (Figure adapted from [9])
1.3. Tool for Genome Editing
There are four major tools capable of inducing site-specific double strand breaks (DSB); zinc
finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease and
CRISPR-Cas complex [11]. Any of these platforms may be programmed for DSB-induced genome
editing in prokaryotes and eukaryotes by exploiting their endogenous DNA repair mechanisms [11].
Breaks in the genome are remediated via one of two main repair mechanisms; non-
homologous end-joining (NHEJ) or homology-directed repair (HDR) [11]. The NHEJ pathway does
not utilize a template to flank the break region, hence, there is random insertion and/or deletions
(indels) of nucleotides at the site of damage [11]. This error-prone repair mechanism results in
frameshifts at the break site (Fig. 2.), and if the region encodes for a gene, its function is knocked out
[11]. On the other hand, HDR employs a template sequence that is homologous to the break site [11].
This high fidelity repair mechanism is mainly active during the S and G2 phases of mammalian cell
cycle, and the sister chromatid is used as template for the break sites [11]. However, an exogenous
template may be introduced to flank these break sites to incorporate synthetic sequences (Fig. 2) [11].
The CRISPR-Cas system has been praised for its efficiency, precision, low cost and ease of
use [8]. Furthermore, for multiplexing purposes, this system has the ability to simultaneously edit
multiple target sites without the need of cumbersome protein engineering as required in ZFNs or
TALENs [7][8]. Three types of CRISPR systems have been identified, however, only the type II
CRISPR-Cas system is the least complex as it only requires three components to function; Cas
endonuclease, mature crRNA and auxiliary tracrRNA [2][7]. This system has been further simplified
by fusing the crRNA with the tracrRNA to form a single guide RNA (sgRNA) [7][11].