ABSTRACT Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short

palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its

guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation

of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs.

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CRISPR-CAS12A NUCLEASES FUNCTION WITH STRUCTURALLY ENGINEERED CRRNAS: SYNTHETIC TRACRRNA Article Open access 16 July 2022 EFFICIENT ENGINEERING OF HUMAN AND MOUSE PRIMARY CELLS USING

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ACKNOWLEDGEMENTS We thank B. Holmes for help with computational tools. P.D.H. is a James Mills Pierce Fellow and D.A.S. is a National Science Foundation (NSF) pre-doctoral fellow. V.A. is

supported by NIH Training Grants T32GM007753 and T32GM008313. This work was supported by an NIH Director's Pioneer Award (1DP1-MH100706); an NIH Transformative R01 grant

(1R01-DK097768); the Keck, McKnight, Damon Runyon, Searle Scholars, Vallee, Merkin, Klingenstein and Simons Foundations; Bob Metcalfe; and Jane Pauley. Reagents are available to the academic

community through Addgene and associated protocols; support forums and computational tools are available via the Zhang lab website (http://www.genome-engineering.org/). AUTHOR INFORMATION

Author notes * F Ann Ran and Patrick D Hsu: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Broad Institute of Massachusetts Institute of Technology (MIT) and

Harvard, Cambridge, Massachusetts, USA F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott & Feng Zhang * McGovern Institute for Brain Research, Cambridge,

Massachusetts, USA F Ann Ran, Patrick D Hsu, David A Scott & Feng Zhang * Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA., F Ann Ran, Patrick D Hsu, David

A Scott & Feng Zhang * Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA F Ann Ran, Patrick D Hsu, David A Scott & Feng Zhang * Department of Molecular and

Cellular Biology, Harvard University, Cambridge, Massachusetts, USA F Ann Ran & Patrick D Hsu * Program in Biophysics, Harvard University, MIT, Cambridge, Massachusetts, USA Vineeta

Agarwala * Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA Vineeta Agarwala Authors * F Ann Ran View author publications You can also search for

this author inPubMed Google Scholar * Patrick D Hsu View author publications You can also search for this author inPubMed Google Scholar * Jason Wright View author publications You can also

search for this author inPubMed Google Scholar * Vineeta Agarwala View author publications You can also search for this author inPubMed Google Scholar * David A Scott View author

publications You can also search for this author inPubMed Google Scholar * Feng Zhang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

F.A.R., P.D.H., J.W., D.A.S. and F.Z. designed and performed the experiments. V.A. contributed to the online tool. F.A.R., P.D.H. and F.Z. wrote the manuscript with help from all authors.

CORRESPONDING AUTHOR Correspondence to Feng Zhang. ETHICS DECLARATIONS COMPETING INTERESTS A patent application has been filed relating to this work. SUPPLEMENTARY INFORMATION SUPPLEMENTARY

DATA 1 Supplementary sequences (PDF 97 kb) SUPPLEMENTARY DATA 2 pSpCas9(BB) plasmid sequence (TXT 12 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Ran, F., Hsu, P., Wright, J. _et al._ Genome engineering using the CRISPR-Cas9 system. _Nat Protoc_ 8, 2281–2308 (2013). https://doi.org/10.1038/nprot.2013.143 Download citation * Published:

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