The CRISPR-Cas9 system, which simply uses a guide RNA and Cas9 nuclease to identify and cut target DNA sequences, comprises a robust technology that has been used in diverse and innovative applications in biology. In the original bacterial system, the chimeric CRISPR RNAs contain a "guide" sequence homologous to the genome of the invading pathogen. The system works by Cas9 unfolding and running along the host DNA until reaching a region complementary to the CRISPR guide sequence, which then binds to and cleaves the target site by Watson-Crick base pairing and the endonuclease activity of the Cas9 protein. This system has been adapted to introduce a genetic manipulation in cells by designing CRISPR "guide" sequences from the organisms own genome.
The CRISPR-Cas9 system has incomparable advantages over other gene editing tools. For example, the CRISPR-Cas9 system has more target sites than ZFNs and TALENs, and Cas9 has many variants that can be used in a variety of studies. Moreover, the system is extremely easy to use and can be executed in almost any laboratory. CRISPR-Cas9-based tools have greatly enhanced our ability to perform systematic analyses of gene function, as well as to reproduce tumor-associated chromosomal translocations precisely. This technology has also paved the way for the dissection of redundant gene functions, epigenetics and possible gene therapy.
The CRISPR story began in 1987. While studying the iap enzyme involved in isozyme conversion of alkaline phosphatase in Escherichia coli, Nakata and colleagues reported a curious set of 29nt repeats downstream of the iap gene. Unlike most repetitive elements, which typically take the form of tandem repeats like TALE repeat monomers, these 29nt repeats were interspaced by five intervening 32nt nonrepetitive sequences. Over the next ten years, as more microbial genomes were sequenced, additional repeat elements were reported from genomes of different bacterial and archaeal strains. Mojica and colleagues eventually classified interspaced repeat sequences as a unique family of clustered repeat elements present in more than 40% of bacteria and 90% of archaea.
In 2002, these short repeats were ofﬁcially named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In the next few years, a series of Cas genes, situated next to the CRISPR locus, were identiﬁed in CRISPR-containing prokaryotes. Subsequently, with the discovery of the Cas protein, protospacer adjacent motif (PAM), CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), many details regarding CRISPR systems were quickly unveiled. In 2007, after further analysis and comparison, it was conﬁrmed that CRISPR systems were a kind of bacterial adaptive immune defense mechanism that protected bacteria against plasmids and phages. The unique protein of the type II CRISPR system is Cas9, which was conﬁrmed to be an essential component of the CRISPR systems.
First, Moineau and colleagues used genetic studies in Streptococcus thermophilus to reveal that Cas9 is the only enzyme within the cas gene cluster that mediates target DNA cleavage. Next, Charpentier and colleagues revealed a key component in the biogenesis and processing of crRNA in type II CRISPR systems-a noncoding tracrRNA that hybridizes with crRNA to facilitate RNA-guided targeting of Cas9. This dual RNA hybrid, together with Cas9 and endogenous RNase III, is required for processing the CRISPR array transcript into mature crRNAs. Heterologous expression of mature crRNA-tracrRNA hybrids as well as sgRNAs directs Cas9 cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated gene editing. Multiple guide RNAs can also be used to target several genes at once.
Subsequently, the CRISPR-Cas9 system has come of age as a novel targeted genome engineering technology and has been successfully used in numerous species. Furthermore, the technique is constantly being modiﬁed and optimized with the aim of achieveing different outcomes, and new studies are continually forthcoming. During the past 30 years, CRISPR has evolved from 'curious sequences of unknown biological function' into a promising genome editing tool that is employed globally.
For example, researches have demonstrated a catalytically inactive Cas9 protein (dCas9) lacking endonuclease activity that can be used as a platform for RNA-guided transcription regulation; this modified system is called CRISPR interference (CRISPRi). Unlike RNAi-based silencing, the CRISPR-dCas9 system directly blocks transcription elongation within protein-coding regions and leads to dramatic suppression of transcription, with no detectable off-target effects. Both repressive and activating effector domains, such as KRAB and VP64, can be fused to dCas9 to either repress (CRISPRi) or activate (CRISPRa) the transcription of target genes. This CRISPRi/a system was further modified to control the transcription levels of endogenous genes up to ~1000-fold, allowing researchers to determine how gene dosage affects cellular functions of interest.
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