RNA-Guided Gene Editing

RESEARCH HIGHLIGHTS GENETICS RNA-guided gene editing Chr. 3 Targeted gene modification can be guid- adjacent motif (PAM), which takes the CCR5 ed by programmable RNA in bacteria, form NGG, immediately following the zebrafish and mammalian cells. 20-base-pair target sequence. Humble creatures, prokaryotes and In contrast to current tools, zinc-finger 123 5ʹ- -3ʹ viruses, have an illustrious history of pro- nucleases and transcription activator–like 3ʹ- -5ʹ viding biologists with molecular tools. effector nucleases (TALENs), targeting of 5ʹ- Chimeric RNA Where, after all, would molecular biol- a new sequence with CRISPR-Cas requires 3ʹ- ogy—or for that matter, all of genomics— the design of only a new RNA guide and Cas9 be without restriction endonucleases or not a new pair of enzymes. The efficiency of DNA polymerases? And, as a recent flurry cleavage of mammalian genes reported in RNA-guided editing. The schematic depicts Cas9 of papers reporting RNA-guided genome these studies is equivalent to or better than (green) cleavage of a target site in the CCR5 engineering demonstrates, the bacteria are that seen with TALENs. The Church and gene, mediated by a chimeric guide RNA. Image adapted from Nature Biotechnology. still at it. Zhang groups report mutations induced by The clustered, regularly interspaced, nonhomologous end joining–based repair short palindromic repeats (CRISPR) at the cleavage site as well as sequence ing, to Escherichia coli (Jiang et al., 2013). system is a component of bacterial introduction by homologous recombina- As part of this work, the researchers stud- and archaeal immunity. The CRISPR- tion from a donor construct (Mali et al., ied the sequence requirements for target associated (Cas) endonuclease cleaves 2013; Cong et al., 2013). The Kim group cleavage. foreign nucleic acids, directed to its target derived clonal mutant cell lines by limiting They found that single nucleotide sequence by two small RNAs. In work pub- dilution and without antibiotic selection, changes at every position in the 12 PAM- lished last year, researchers showed that the which suggests that the system can be well proximal nucleotides of the target sequence CRISPR-Cas system of Streptococcus pyo- tolerated by cells (Cho et al., 2013). can abrogate cleavage, but not every change genes could be programmed to direct in The group of Keith Joung and col- does so to the same degree. And though the © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature vitro cleavage of desired target sequences. leagues, also at Harvard Medical School, PAM sequence is critical for cleavage, some This raised the possibility that the have applied RNA-guided editing to single-nucleotide changes are tolerated approach could be used for targeted gene mutate endogenous genes in the zebrafish (albeit with reduced cleavage). Although npg editing in vivo. With an almost startling (Hwang et al., 2013), achieving mutation these experiments were conducted in bac- rapidity, five recently published papers at >80% of targeted sites. Although they teria and results from the other groups in now show that it can. report no major toxic effects on embryos, the eukaryotic context and in vitro suggest The groups of George Church at Harvard phenotypes in adult fish and transmission that RGENs are specific, this is an area that Medical School, Feng Zhang at the Broad of the mutations to the next generation will benefit from more examination. Institute and Jin-Soo Kim at Seoul National remain to be examined. What remains eminently clear is that University describe methodological varia- The fact that clonal cell lines can be prokaryote biology is still fertile hunting tions on essentially the same theme: the derived and that there is no unexpected ground for new laboratory methods. S. pyogenes Cas9 nuclease, together with toxicity in the fish embryo suggests that Natalie de Souza two noncoding RNAs (the CRISPR RNA RNA-guided nucleases (RGENs) do not RESEARCH PAPERS (crRNA) and trans-activating crRNA cleave indiscriminately in eukaryotic Cho, S.W. et al. Targeted genome engineering in human (tracrRNA)), effects the cleavage of endog- cells. But what about their finer speci- cells with the Cas9 RNA-guided endonuclease. Nat. enous sequences in mammalian cells. ficity? How likely are RGENs to cut at Biotechnol. advance online publication (29 January 2013). The two RNAs can be provided either related off-target sites? A fifth study from Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science published online (3 in chimeric form or as two separate mole- Luciano Marraffini and colleagues at The January 2013). cules; they may be transcribed from a plas- Rockefeller University offers some sober- Hwang, W.Y. et al. Efficient genome editing in mid or transcribed in vitro prior to delivery ing observations. zebrafish using a CRISPR-Cas system. Nat. Biotechnol. into the cell. Importantly, in all cases, the Marraffini and colleagues use the advance online publication (29 January 2013). Jiang, W. et al. RNA-guided editing of bacterial target sequence is specified by comple- CRISPR system for RNA-guided editing genomes using CRISPR-Cas systems. Nat. Biotechnol. mentarity to the 20-base-pair crRNA. The of bacterial genomes, applying this both advance online publication (29 January 2013). minimal constraint on the sequence is the to the recombinogenic Streptococcus pneu- Mali, P. et al. RNA-guided human genome engineering requirement for the so-called protospacer moniae and, in tandem with recombineer- via Cas9. Science published online (3 January 2013). NATURE METHODS | VOL.10 NO.3 | MARCH 2013 | 189.

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