PMID- 29512652
OWN - NLM
STAT- MEDLINE
DCOM- 20180817
LR  - 20240104
IS  - 1476-4687 (Electronic)
IS  - 0028-0836 (Print)
IS  - 0028-0836 (Linking)
VI  - 556
IP  - 7699
DP  - 2018 Apr 5
TI  - Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.
PG  - 57-63
LID - 10.1038/nature26155 [doi]
AB  - A key limitation of the use of the CRISPR-Cas9 system for genome editing and 
      other applications is the requirement that a protospacer adjacent motif (PAM) be 
      present at the target site. For the most commonly used Cas9 from Streptococcus 
      pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered 
      Cas9 variants that have been shown to function efficiently in mammalian cells 
      offer a PAM less restrictive than NGG. Here we use phage-assisted continuous 
      evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a 
      broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of 
      xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are 
      active in mammalian cells, and supports applications in human cells including 
      targeted transcriptional activation, nuclease-mediated gene disruption, and 
      cytidine and adenine base editing. Notably, despite its broadened PAM 
      compatibility, xCas9 has much greater DNA specificity than SpCas9, with 
      substantially lower genome-wide off-target activity at all NGG target sites 
      tested, as well as minimal off-target activity when targeting genomic sites with 
      non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and 
      establish that there is no necessary trade-off between Cas9 editing efficiency, 
      PAM compatibility and DNA specificity.
FAU - Hu, Johnny H
AU  - Hu JH
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Miller, Shannon M
AU  - Miller SM
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Geurts, Maarten H
AU  - Geurts MH
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Tang, Weixin
AU  - Tang W
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Chen, Liwei
AU  - Chen L
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Sun, Ning
AU  - Sun N
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Zeina, Christina M
AU  - Zeina CM
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Gao, Xue
AU  - Gao X
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Rees, Holly A
AU  - Rees HA
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Lin, Zhi
AU  - Lin Z
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
FAU - Liu, David R
AU  - Liu DR
AD  - Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of 
      MIT and Harvard, Cambridge, Massachusetts 02142, USA.
AD  - Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 
      02138, USA.
AD  - Department of Chemistry and Chemical Biology, Harvard University, Cambridge, 
      Massachusetts 02138, USA.
LA  - eng
GR  - HHMI_/Howard Hughes Medical Institute/United States
GR  - R01 EB022376/EB/NIBIB NIH HHS/United States
GR  - R35 GM118062/GM/NIGMS NIH HHS/United States
GR  - RM1 HG009490/HG/NHGRI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20180228
PL  - England
TA  - Nature
JT  - Nature
JID - 0410462
RN  - 0 (CRISPR-Associated Proteins)
RN  - 9007-49-2 (DNA)
RN  - EC 3.1.- (Deoxyribonucleases)
SB  - IM
CIN - Nat Rev Genet. 2018 May;19(5):250-251. PMID: 29527014
MH  - CRISPR-Associated Proteins/*genetics/*metabolism
MH  - CRISPR-Cas Systems/*genetics
MH  - DNA/*genetics/*metabolism
MH  - DNA Cleavage
MH  - Deoxyribonucleases/metabolism
MH  - Directed Molecular Evolution
MH  - Gene Editing/*methods
MH  - Genome, Human/genetics
MH  - HEK293 Cells
MH  - Humans
MH  - *Mutation
MH  - Nucleotide Motifs
MH  - Streptococcus pyogenes/enzymology/genetics
MH  - Substrate Specificity/*genetics
MH  - Transcriptional Activation
PMC - PMC5951633
MID - NIHMS945381
COIS- The authors declare competing financial interests: J.H.H. and D.R.L. have filed 
      patent applications on this work. D.R.L. is a consultant and co-founder of Editas 
      Medicine, Beam Therapeutics, and Pairwise Plants, companies that use genome 
      editing technologies. The authors declare no competing non-financial interests.
EDAT- 2018/03/08 06:00
MHDA- 2018/08/18 06:00
PMCR- 2018/08/28
CRDT- 2018/03/08 06:00
PHST- 2018/01/17 00:00 [received]
PHST- 2018/02/21 00:00 [accepted]
PHST- 2018/03/08 06:00 [pubmed]
PHST- 2018/08/18 06:00 [medline]
PHST- 2018/03/08 06:00 [entrez]
PHST- 2018/08/28 00:00 [pmc-release]
AID - nature26155 [pii]
AID - 10.1038/nature26155 [doi]
PST - ppublish
SO  - Nature. 2018 Apr 5;556(7699):57-63. doi: 10.1038/nature26155. Epub 2018 Feb 28.