RESEARCH HIGHLIGHTS

GENOMICS New kid on the CRISPR block

The small, single RNA–guided nuclease and direct it to its DNA target next to per PAM. Outfitted with a nuclear localiza- Cpf1 is active in human cells. a G-rich protospacer-adjacent motif (PAM). tion signal, each Cpf1, together with a guide Chances are that you have not heard of Zhang and his team set out to character- RNA targeting DNMT1, was transfected into Francisella novicida, a Gram-negative patho- ize another CRISPR-Cas system that had human cells. Two showed efficient cleavage genic bacterium. Yet its defense system could recently been identified in several bacterial in multiple different cell lines. be the basis for improved genome engineer- genomes, the 1.3-kb Cpf1 (CRISPR from Cpf1’s small size and staggered cutting ing, as Feng Zhang and his team from the Prevotella and Francisella). Working with may allow for easy delivery to cells and effec- of MIT and Harvard recently Cpf1 from F. novicida in Escherichia coli, tive integration of DNA inserts in their prop- demonstrated. they demonstrated three advantages in er orientation. The fact that Cpf1 cleaves dis- In recent years, the bacterial adaptive addition to its small size: it does not require tant from its PAM could allow for multiple immune system CRISPR-Cas9, in which a tracrRNA and is recruited and targeted by rounds of targeting with the same guide RNA guides the Cas9 nuclease to its comple- a single guide RNA around 44 nucleotides in RNA. For example, Zhang’s team envisions mentary DNA, has been applied to genome length; it cleaves about 20 bases away from the introduction of an insertion or deletion modification in many species. Most of this a T-rich PAM, rather than adjacent to the in round one and homologous recombina- work is based on Cas9 from Streptococcus PAM as Cas9 does; and it produces DNA tion in round two. Once issues such as off- pyogenes (SpCas9), which, despite its suc- double-strand breaks with several bases of target cleavage and efficacy are addressed, cessful use in this system, has some limita- 5ʹ overhang, in contrast to Cas9’s blunt ends. Cpf1 will be a welcome complement to Cas9. tions. The SpCas9 protein is large, encoded The big question was whether Cpf1 would Nicole Rusk by a >4-kilobase gene, and it requires the be active in higher organisms. The team RESEARCH PAPERS combination of an ~55-nucleotide trans- looked at Cpf1 proteins from 16 species and Zetsche, B. et al. Cpf1 is a single RNA-guided activating CRISPR RNA (tracrRNA) and determined the PAM sequences of seven of endonuclease of a class 2 CRISPR-Cas system. Cell an ~45-nucleotide guide RNA to recruit them, which varied only in the number of T’s 163, 759–771 (2015). America, Inc. All rights reserved. America, Inc. © 201 5 Nature npg

NATURE METHODS | VOL.12 NO.12 | DECEMBER 2015 | 1117