The CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein-9 nuclease) includes the RNA-guided Cas9 nuclease, which binds to specific DNA sequences (complementary to the RNA-guide sequence) and creates double-stranded breaks (DSBs) on the DNA. The dsDNA breaks can be repaired via homology-directed repair (HDR) or nonhomologous end-joining (NHEJ). Currently, CRISPR-Cas9 system is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. This system has been adapted to introduce a genetic manipulation in mammalian cells by designing CRISPR "guide" sequences from the organisms own genome. As a new revolutionary genome-editing tool, CRISPR-Cas9 system is opening new avenues for gene engineering.
The CRISPR-Cas9 system, which is derived from a prokaryotic adaptive immune system, is composed of two core components: the RNA-guided DNA endonuclease Cas9 and a chimeric single guide RNA (sgRNA). The sgRNA, which has an invariant scaffold region and a spacer region, is derived from CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The sgRNA binds to Cas9 and directs it to the locus of interest by a 20-nt guide sequence via base pairing to the genomic target. The target sequence in genomic DNA paired to sgRNA sequence is immediately followed by either an NGG or NAG trinucleotide for S. pyogenes-derived Cas9 (other Cas9 orthologues recognize different PAM), called the protospacer adjacent motif (PAM). The PAM is located on the immediate 3′ end of the sgRNA recognition sequence, but it is not a part of the 20-nt guide sequence within sgRNA.
Fig 1. Schematic representation of the domain organization of Type II-A Streptococcus pyogenes Cas9 (SpyCas9, or SpCas9).
The nuclease Cas9 consists of two catalytic active domains: HNH and RuvC. The HNH domain is a single nuclease domain, whereas the RuvC domain contains three subdomains across the linear protein sequence. The HNH and RuvC domains are properly positioned for cleavage of the complementary and non-complementary strands of the target DNA, respectively. Permanent CRISPR-Cas9-mediated modification of single or multiple endogenous loci can be achieved via transient or stable delivery of CRISPR-Cas9 system components. Several groups have reported successful editing of endogenous genes in cells in culture via transient transfection of plasmid DNA encoding Cas9 and sgRNAs or Cas9-sgRNA ribonucleoprotein complexes (RNPs). Alternatively, CRISPR components can be stably delivered into cells through the use of retroviruses or lentiviruses.
Type II CRISPR-Cas9 system employs a single DNA endonuclease, Cas9, to recognize sequence-specific double-stranded DNA (dsDNA) substrates and cleave each strand with a distinct nuclease domain (HNH or RuvC). During this silencing process, an additional small noncoding RNA, called the trans-activating crRNA (tracrRNA), base pairs with the repeat sequence in the crRNA to form a unique dual-RNA hybrid structure. If the genomic DNA is complementary to the sequence of the artificial sgRNA, this dual-RNA guide directs Cas9 to cleave any DNA containing a complementary 20-nucleotide (nt) target sequence and adjacent PAM. In conclusion, the CRISPR-Cas9 system uses Cas9, which complexes with the sgRNA, to cleave DNA 3-4 base pairs upstream of PAM and generates double-strand breaks (DSBs) in a sequence-specific manner.
Fig 1. The mechanism of CRISPR-Cas9-mediated genome engineering
The DSBs are then repaired either by non-homologous end joining (NHEJ)-mediated error-prone DNA repair or homologous directed repair (HDR)-mediated error-free DNA repair. The former repair can rapidly ligate the DSB, but generates small insertion and deletion mutations at the target sites. These mutations could disrupt and abolish the function of target genes or genomic elements. For instance, an sgRNA that targets a protein-coding region can produce loss of function frame-shifting indels through NHEJ-mediated DNA repair. HDR-mediated error-free DNA repair requires a homology-containing donor DNA sequence as repair template. Numerous studies published over the last few years have demonstrated efficient gene disruption and gene modification in a variety of cells and organisms via CRISPR-Cas9-mediated NHEJ or HDR, respectively.
Achieving site-specific DNA recognition and cleavage requires that Cas9 be assembled with guide RNA (a native crRNA-tracrRNA or an sgRNA) to form an active DNA surveillance complex. The 20-nt spacer sequence of crRNA confers DNA target specificity, and the tracrRNA plays a crucial role in Cas9 recruitment. Genetic and biochemical experiments have defined the role of a so-called seed sequence of RNA nucleotides within the spacer region of crRNAs that is particularly important for target specificity. In type II CRISPR systems, the seed region has been defined as the PAM-proximal 10-12nt located in the 3' end of the 20-nt spacer sequence. Mismatches in this seed region severely impair or completely abrogate target DNA binding and cleavage, whereas close homology in the seed region often leads to off-target binding events even with many mismatches elsewhere.
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