Format

Send to

Choose Destination
Nat Biotechnol. 2019 Mar;37(3):276-282. doi: 10.1038/s41587-018-0011-0. Epub 2019 Feb 11.

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Kleinstiver BP1,2,3,4,5, Sousa AA1,2,3, Walton RT1,2,3,5, Tak YE1,2,3,4, Hsu JY1,2,3,6, Clement K1,2,4,7, Welch MM1,2,3, Horng JE1,2,3, Malagon-Lopez J1,2,3,4,8,9, Scarfò I2,10,11, Maus MV2,10,11, Pinello L1,2,4,7, Aryee MJ1,2,4,7,8, Joung JK12,13,14,15.

Author information

1
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
2
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.
3
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
4
Department of Pathology, Harvard Medical School, Boston, MA, USA.
5
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
6
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
7
Cell Circuits and Epigenomics Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
8
Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA.
9
Advance Artificial Intelligence Research Laboratory, WuXi NextCODE, Cambridge, MA, USA.
10
Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, MA, USA.
11
Harvard Medical School, Boston, MA, USA.
12
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
13
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
14
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
15
Department of Pathology, Harvard Medical School, Boston, MA, USA. jjoung@mgh.harvard.edu.

Abstract

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.

PMID:
30742127
PMCID:
PMC6401248
[Available on 2019-08-11]
DOI:
10.1038/s41587-018-0011-0
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Nature Publishing Group
Loading ...
Support Center