alexa The CRISPR-Cas9 System

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The CRISPR-Cas9 System

 

In bacteria and archaea, CRISPR loci usually consist of three components: a cluster of cas genes and two non-coding RNA elements, trans-activating CRISPR RNA (trascrRNA) and a characteristic array consisting of repetitive sequences flanking unique spacer sequences (Figure 1A). Each spacer is derived from invading phage or plasmid DNA. Transcription of the array yields individual CRISPR RNAs (crRNAs, consisting of spacer-repeat fragments), which localizes the crRNA: tracrRNA: Cas9 complex to target DNA where the effector Cas9 nuclease cuts both strands of DNA (double-strand breaks, DSBs) that matches the crRNA, and consequently, leads to the inactivation of invading DNA [2-4]. In mammalian and other cells, CRISPR-Cas induced DSBs can be repaired through two endogenous mechanisms: the non-homologous end joining (NHEJ) method is generally used for the creation of a frameshift deleterious mutation, while the homology directed repair (HDR) is preferred for the introduction of a specific point mutation or addition of genes of interest. This precision targeting feature of the CRISPR-Cas9 system is of great interest for the study of biological processes [5].
 
What makes the CRISPR-Cas9 system even more attractive is the ease, high efficiency, and versatility of the technology. To simplify the CRISPR-Cas9 system, Jinek M et al. synthesized a single RNA chimera of dual-tracrRNA:crRNA (single-guide RNA, sgRNA), and successfully used it to direct sequence specific Cas9 double-strand DNA cleavage in a test tube in 2012 [6]. This study suggested the potential application of the CRISPR-Cas9 system for RNA-programmable genome engineering. In February 2013, two groups simultaneously demonstrated that the RNA-guided CRISPR-Cas9 system functions in both human and mouse cells and that multiplex editing of target genes is feasible upon introduction of multiple sgRNAs at the same time [6,7]. Shortly after these two milestone papers were published in Science, the CRISPR-Cas9 system was successfully used for genome modifications in other organisms such as plants [8-10], Caenorhabditis elegans [11-13], Drosophila [14,15], Zebrafish [10,15-17], and Xenopus tropicalis [18,19], suggesting that the CRISPR-Cas9 system may have broad applications in the biomedical sciences.
 
More recently, the CRISPR-Cas9 system was modified to create a more efficient, one-step, gene targeting technology. By co-injecting Cas9 mRNA and sgRNAs of interest into cells, Dr. Zhang’s group was able to simultaneously target five genes in mouse embryonic stem cells, and mice generated from zygotes co-injected with Cas9 mRNA and sgRNAs targeting Tet1 and Tet2 were shown to carry biallelic mutations in both genes with an efficiency of 80% [20]. This approach has much higher mutation efficiency and a much lower rate of offtarget effects than the zinc-finger nuclease technique [21]. Similarly, reporter and conditional mutant mice were generated by this one-step co-injection of zygotes with Cas9 mRNA and different sgRNAs as well as DNA vectors [22]. Traditional generation of mice with multiple gene mutations requires careful breeding over many generations and may take 1-2 years. Therefore, this one-step approach to generating animals carrying mutations in multiple genes will greatly accelerate the in vivo study of gene functions and gene-gene interactions. Given that the CRISPR-Cas9 system’s sgRNAs are now much easier to make than proteins exploited in zinc finger and TALEN genome engineering technologies [23], it is possible to target virtually any gene using the CRISPR-Cas9 system, and a genome-wide resource of unique sgRNAs that target human exons is now available [7].

Citation: Yan Y, Wei D (2013) The CRISPR-Cas9 System: A Powerful Tool for Genome Engineering and Regulation. Adv Genet Eng 2:e103. doi: 10.4172/2169-0111.1000e103

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