Research
CRISPR-mediated RNA-guided endonucleasesIn January 2013, several groups (Cho et al, Nat. Biotechnol. 2013; Cong et al, Science 2013; Mali et al, Science 2013; Hwang et al, Nat. Biotechnol. 2013; Jiang et al, Nat. Biotechnol. 2013; Jinek et al, Elife 2013) independently reported a new class of genome editing nucleases — termed RNA-guided engineered nucleases (RGENs) herein to avoid confusion with the original type II clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) adaptive immune system in bacteria — the specificity of which is mostly determined by small guide RNAs rather than by DNA-binding proteins. They cleave chromosomal DNA in a site-specific manner, which triggers endogenous DNA repair systems that result in targeted genome modification.
An RGEN is comprised of CRISPR-associated protein (Cas9), a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), which form the dualRNA–Cas9. Alternatively, an RGEN can contain Cas9 and a single-chain guide RNA (sgRNA). The guide sequence in the crRNA (fig a) or sgRNA (fig b) is complementary to a 20bp target DNA sequence known as protospacer, which is next to the 5'-NGG-3' (where N represents any nucleotide) sequence known as protospacer adjacent motif (PAM) (Kim et al, Nat. Rev. Genet. 2014).
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