Engineered Cpf1 variants with altered PAM specificities
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Abstract
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.
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AsCas12a has different PAM requirements and natural very high specificity.
“Among these, AsCas12a has been widely used for its different PAM requirements and a natural very high specificity17–20.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [17]
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Self-citation
- Authors
- Gao, L., Cox, D. B. T., Yan, W. X., Manteiga, J. C., Schneider, M. W., Yamano, T., Nishimasu, H., Nureki, O., Crosetto, N., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Massachusetts Institute of Technology (US)
- The University of Tokyo (JP)
- Japan Science and Technology Agency (JP)
- Science for Life Laboratory (SE)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2017-06-05
- Volume
- 35
- Issue
- 8
- Pages
- 789-792
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC5548640
- OpenAlex
- W2621786780
- Cited by
- 460
- References
- 7
- Citations by year
- 2026: 29 · 2025: 35 · 2024: 41 · 2023: 45 · 2022: 56 · 2021: 65 · 2020: 62 · 2019: 62 · 2018: 41 · 2017: 22 · 2016: 1
- FWCI
- 21.01
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- PMC
- Funders
- National Science Foundation (T32GM007753)
- Damon Runyon Cancer Research Foundation
- Japan Agency for Medical Research and Development
- Broad Institute
- Ministry of Education, Culture, Sports, Science and Technology (26291010, 15H01463)
- Karolinska Institutet
- Vetenskapsrådet (521-2014-2866)
- National Institutes of Health
- Japan Society for the Promotion of Science (26291010, 15H01463)
- National Institute of Mental Health (DP1MH100706, 5DP1-MH100706, R01MH110049)
- National Institute of General Medical Sciences (T32GM007753, T32GM007287)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097768)
- Precursory Research for Embryonic Science and Technology (JPMJPR13L8)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas12a, protospacer adjacent motif, structure-guided mutagenesis, RNA-guided endonuclease, genome editing, off-target effects, bliss
- MeSH terms
- Bacterial Proteins, Endonucleases, Genetic Engineering, Humans, Genetic Variation, Mutagenesis, Site-Directed, Acidaminococcus, HEK293 Cells
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