Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells
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Abstract
The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9). We also report that four to six bases at the 3' end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.
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GUIDE-seq off-target profiling of AsCas12a-crRNA+11 in HEK293/3272-26A>G cells showed exclusive editing of the 3272-26A>G CFTR locus, with no detectable non-specific cleavages in the second allele or any other genomic loci.
“Off-target profiling of AsCas12a-crRNA+11 genome editing in HEK293/3272-26A>G cells18,42 showed very high specificity, as demonstrated by exclusive editing of the 3272-26A>G CFTR locus, while non-specific cleavages in the second allele, or any other genomic loci, could not be detected (Fig. 2c and Supplementary Fig. 4c).”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [18]
The Results report GUIDE-seq profiling of AsCpf1 in HEK293 cells and no detected off-target sites for one tested crRNA. They do not report profiling AsCas12a-crRNA+11 in HEK293/3272-26A>G cells, editing of the CFTR locus, or cleavage testing of its second allele. The reported general specificity does not establish the claimed result for that guide and cell line. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe high-fidelity AsCas12a genetic repair applied in this study to CFTR splicing defects resulted in allele-specific and genome-wide off-target-free editing.
“The high-fidelity AsCas12a genetic repair18 applied in this study to CFTR splicing defects, resulted in an allele specific and genome-wide off-target free editing.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [18]
The Discussion (page not provided) reports that AsCpf1 is highly specific in human cells and suggests it might be suitable for allele-specific editing. It does not report CFTR splicing-defect repair or demonstrate allele-specific editing in that setting. Its finding that most tested nucleases showed no detectable off-target effects does not establish genome-wide off-target-free editing for the repair described in the citing sentence. (LLM, conf=0.88)
Experimental — accuracy not yet measuredAsCas12a 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 [18]
The authors developed a genome editing strategy using AsCas12a that permanently corrects CFTR splicing defects of at least two relevant splicing mutations (3272-26A>G and 3849+10kbC>T) in combination with a single crRNA.
“In our study, we developed a genome editing strategy using one of the most precise programmable nucleases18, AsCas12a, that permanently corrects CFTR splicing defects of at least two relevant splicing mutations (3272-26A>G and 3849+10kbC>T) in combination with a single crRNA.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [18]
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- Authors
- Kleinstiver, B. P., Tsai, S. Q., Prew, M. S., Nguyen, N. T., Welch, M. M., Lopez, J. M., McCaw, Z. R., Aryee, M. J., & Joung, J. K.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Corresponding author
- Kleinstiver, Benjamin P
- Joung, J Keith
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2016-06-27
- Volume
- 34
- Issue
- 8
- Pages
- 869-874
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4980201
- OpenAlex
- W2465120080
- Cited by
- 670
- References
- 10
- Citations by year
- 2026: 30 · 2025: 44 · 2024: 52 · 2023: 62 · 2022: 71 · 2021: 77 · 2020: 85 · 2019: 77 · 2018: 71 · 2017: 79 · 2016: 18
- FWCI
- 42.46
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Nature Biotechnology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Institutes of Health (NIH-R01)
- Natural Sciences and Engineering Research Council of Canada
- National Institute of General Medical Sciences (DP1GM105378, R01GM107427)
- 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
- off-target cleavage, GUIDE-seq, human cells, genome editing
- MeSH terms
- Bacterial Proteins, Binding Sites, Chromosome Mapping, Endonucleases, Enzyme Activation, Humans, Protein Binding, Substrate Specificity, Genome, Human, Base Pair Mismatch, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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