Plant Genome Editing Using FnCpf1 and LbCpf1 Nucleases at Redefined and Altered PAM Sites

摘要

Dear Editor, CRISPR from Prevotella and Francisella 1 (Cpf1) is an emerging RNA-guided endonuclease system that relies on thymidine-rich protospacer adjacent motif (PAM) for DNA targeting (Zetsche et al.,2015).CRISPR-Cpf1 has unique features that could be advantageous over the CRISPR-Cas9 system.For example,Cpf1 requires only a 42 nt crRNA,while Cas9 uses ～100 nt gRNA.While Cas9 generates blunt ends of DNA breaks,the Cpf1 cleavage results in 5'overhangs distal from the protospacer,which may improve efficiency for NHEJ-based gene insertion.Interestingly,Cpf1 proteins also have RNase activity (Fonfara et al.,2016),which was utilized to process crRNA arrays for multiplexed genome editing in both mammalian systems and plants (Wang et al.,2017;Zetsche et al.,2017).Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) and Francisella novicida Cpf1 (FnCpf1) have been shown to work in plants (Endo et al.,2016;Xu et al.,2016;Begemann et al.,2017;Hu et al.,2017;Kim et al.,2017;Tang et al.,2017).Notably,the LbCpf1 system developed by our group achieved nearly 100％ biallelic mutagenesis in rice (Tang et al.,2017).