Cas9 gRNA engineering for genome editing, activation and repression.
We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Samira
S
Kiani
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.|Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Alejandro
A
Chavez
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
Marcelle
M
Tuttle
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Richard N
RN
Hall
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Raj
R
Chari
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Dmitry
D
Ter-Ovanesyan
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Jason
J
Qian
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
Benjamin W
BW
Pruitt
Raytheon BBN Technologies, Cambridge, Massachusetts, USA.
Jacob
http://orcid.org/0000-0002-1663-5102
J
Beal
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Suhani
http://orcid.org/0000-0003-4618-2017
S
Vora
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
Joanna
J
Buchthal
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
Emma J K
EJ
Kowal
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Center for Emergent Behaviors of Integrated Cellular Systems (EBICS), Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Mohammad R
MR
Ebrahimkhani
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.|Institute for Medical Engineering &Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.|Harvard-MIT Program in Health Sciences and Technology, Cambridge, Massachusetts, USA.
James J
JJ
Collins
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.|Center for Emergent Behaviors of Integrated Cellular Systems (EBICS), Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Ron
R
Weiss
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
George
G
Church
D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D017353:Gene Deletion; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D009154:Mutation; D017393:RNA Editing; D014158:Transcription, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems
D064130:CRISPR-Associated Proteins; D005456:Fluorescent Dyes; D000094704:RNA, Guide, CRISPR-Cas Systems