It is of considerable interest for researchers to site-specifically modify the genome at will. Recently, the advent of the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system has greatly facilitated the advancement of genome engineering. This system was first described in bacteria and archaea as an adaptive immune system against viruses, which occurs in three stages: adaptation, expression and interference (Figure 7) (reviewed in [162]).
Figure 7 CRISPR‐Cas is an adaptive immune system in many bacteria and archaea
CRISPR‐Cas immunity occurs in three stages. During the adaptation stage, adaption Cas nucleases complex captures a fragment of the foreign DNA (green line) and integrates it into the CRISPR array between two direct repeats (red triangles) as a new spacer sequence (green hexagon). During the expression stage, the CRISPR array is transcribed and then processed into small CRISPR RNAs (crRNAs). Eventually, crRNA‐guided Cas nucleases are responsible for the cleavage of foreign DNA at the specific sites complementary to the crRNA spacer sequence.
Adapted from [162].
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Once bacteria and archaea are infected, a fragment of the foreign DNA is first captured and integrated into the CRISPR array between two direct repeats as a new spacer sequence, which is executed by certain Cas nucleases exclusively involved in the adaptation stage. The spacer acquisition allows bacteria and archaea to memorize the infection of the respective virus and to transmit this information to the next generation. During the expression stage, the CRISPR array is transcribed and then processed into small CRISPR RNAs (crRNAs). Eventually, crRNAs guide Cas nucleases responsible for the interference stage, to cleave both strands of the foreign DNA specifically at sites that are complementary to the crRNA spacer sequence.
Three different types of the CRISPR-Cas system (I, II and III) are characterized by the molecular mechanism to achieve foreign DNA recognition and cleavage (reviewed in [163]). While type I and type III systems utilize a large complex of Cas proteins for crRNA-guided DNA cleavage, type II system needs only the Cas9 nuclease (Figure 8). Another property of type II system is the requirement of a trans-activating crRNA (tracrRNA). This noncoding RNA hybridizes with crRNA and has been reported not only to be necessary for processing the transcript of the CRISPR array but also to facilitate crRNA-guided DNA cleavage mediated by Cas9 [164]. In addition, the protospacer adjacent motif (PAM), a short sequence motif adjacent to the crRNA-targeted sequence on the foreign DNA, also plays an essential role in type I and type II systems. As the PAM sequence only exists on the foreign DNA and is not integrated into the CRISPR array, it determines the self-nonself discrimination of CRISPR-Cas-mediated DNA cleavage. figure 8 [165]
To date, many studies have accomplished genome engineering in eukaryotes using the system modified from CRISPR-Cas of Streptococcus pyogenes. This bacterium harbors a type II system.
The S. pyogenes Cas9 protein, optimized by codon usage bias and acquisition of nuclear localization, has shown effective nuclease activity within eukaryotic cells [166, 167]. Moreover,
Figure 8 Components of Streptococcus pyogenes type II CRISPR‐Cas9 system
In contrast to type I and type III systems, type II CRISPR‐Cas system requires only one nuclease, Cas9 (light blue), to execute double‐strand DNA cleavage of the target sequence (green). Additionally, tracrRNA (trans‐activating crRNA, red) hybridizes with CRISPR RNA (crRNA, dark green), which facilitates crRNA‐guided DNA cleavage mediated by Cas9. Moreover, PAM (protospacer adjacent motif, orange) determines the self‐nonself discrimination of CRISPR‐Cas‐mediated DNA cleavage. For the application of genome engineering, the tracrRNA:crRNA hybrid has been engineered as a fusion version (gray dotted line), termed single guide RNA (sgRNA). Adapted from [165].
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the tracrRNA:crRNA hybrid has also been engineered as a fusion version, termed single guide RNA (sgRNA), which remains functional to direct sequence-specific double-strand DNA cleavage [168].
The ability of the CRISPR-Cas9 system to recognize specific DNA sequences with subsequent cleavage of both DNA strands makes this system a powerful tool for genome engineering. In mammalian cells, double-strand DNA breaks are predominantly repaired by two different mechanisms: non-homologous end joining (NHEJ) and homology-directed repair (HDR) (Figure 9).
As NHEJ repairs double-strand DNA breaks by blunt end ligation independently of sequence homology, it is an efficient pathway to protect genome integrity throughout the cell cycle.
However, the error-prone mechanism of NHEJ frequently results in insertions or deletions of nucleotides (indels) at the break site [169]. In contrast, owing to the requirement of a homologous DNA template from the sister chromatid, HDR is confined to G2 phase of the cell cycle and executes accurate DNA repair [170]. For the application of CRISPR-Cas9-mediated genome engineering to mammalian cells, indels caused by NHEJ lead to frameshift mutations accompanied by premature stop codons, which enables the knockout of gene expression. Moreover, HDR can be induced by introduction of an additional donor DNA template that is homologous to the DNA sequence targeted by the sgRNA. Thus, genome editing can be achieved by the arrangement of the donor DNA with a desired sequence.
Figure 9 Biology of CRISPR‐Cas9‐mediated genome engineering
Double‐strand DNA breaks caused by CRISPR‐Cas9 system are repaired by HDR (homology‐directed repair, left) or NHEJ (non‐homologous end‐joining, right) in mammalian cells. HDR requires a donor DNA template that is homologous to the DNA sequence targeted by sgRNA. By the arrangement of the donor DNA with the desired sequence, genome editing, including gene insertion or point mutagenesis, is achieved. In contrast, NHEJ repairs double‐strand DNA breaks by blunt end ligation independently of sequence homology. Indels (insertions or deletions) caused by NHEJ lead to frameshift mutations accompanied by premature stop codons, which enables the knockout of gene expression.
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