@article{ee59ed648b1246e6acd43ecfc7bd2179,
title = "Engineered dual selection for directed evolution of SpCas9 PAM specificity",
abstract = "The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.",
author = "Goldberg, {Gregory W.} and Spencer, {Jeffrey M.} and Giganti, {David O.} and Camellato, {Brendan R.} and Neta Agmon and Ichikawa, {David M.} and Boeke, {Jef D.} and Noyes, {Marcus B.}",
note = "Funding Information: We thank Jingchuan Luo, Leslie Mitchell, and other members of the Boeke lab for additional advice on lithium acetate transformations and yeast-related work. We gratefully acknowledge Creative Biogene (Shirley, NY) for performing GUIDE-seq experiments and analyses, NYU Medical Center{\textquoteright}s Research Support Service team for preparation of all glucose-supplemented 2xYT plates and various yeast media used in this work, and the Genome Technology Center for advice on low-complexity amplicon sequencing with the NextSeq 500. We also thank members of the Maurano lab for NextSeq 500 support, flow cell handling, and demultiplexing of raw sequencing data. The plasmids MSP469 and BPK1520, as well as the U2OS.EGFP cell line, were generous gifts from Keith Joung. The expected sequence of the lentiviral donor plasmid originally used during U2OS.EGFP construction was kindly provided by the Cathomen lab. G.W.G. was supported by a NRSA postdoctoral fellowship (F32GM137482) from the NIGMS and a Centers of Excellence in Genomic Science award (RM1HG009491) to J.D.B. from the NHGRI. G.W.G., J.M.S., D.O.G., and D.M.I. were supported by NIGMS awards R01GM118851 and R01GM133936 to M.B.N. Publisher Copyright: {\textcopyright} 2021, The Author(s).",
year = "2021",
month = dec,
day = "1",
doi = "10.1038/s41467-020-20650-x",
language = "English (US)",
volume = "12",
journal = "Nature communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}