RT Journal Article
SR Electronic
T1 Engineered Cpf1 Enzymes with Altered PAM Specificities
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 091611
DO 10.1101/091611
A1 Linyi Gao
A1 David B.T. Cox
A1 Winston X. Yan
A1 John Manteiga
A1 Martin Schneider
A1 Takashi Yamano
A1 Hiroshi Nishimasu
A1 Osamu Nureki
A1 Feng Zhang
YR 2016
UL http://biorxiv.org/content/early/2016/12/04/091611.abstract
AB The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.