Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array
Open accessAbstract
Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.
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Transgene-free plants produced through CRISPR/Cas9 do not contain foreign DNA in the final product, potentially allowing them to bypass GMO regulations.
“Transgene-free plants produced through CRISPR/Cas9 do not contain foreign DNA in the final product, potentially allowing them to bypass GMO regulations (Zetsche et al., 2017; Kim et al., 2017).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Zetsche et al., 2017
The paper’s main section reports CRISPR-Cpf1 editing in mammalian cells and neurons, not the production of transgene-free plants. Searches for plants, foreign DNA, and GMO regulation found no passage addressing whether CRISPR/Cas9-edited plants could bypass regulation. (LLM, conf=0.75)
Experimental — accuracy not yet measuredCRISPR variants Cas12 and Cas13 offered improvements in specificity and reduced off-target effects compared to Cas9.
“The CRISPR variants Cas12 and Cas13 offered improvements in specificity and reduced off-target effects compared to Cas9 and proved to be a highly efficient gene editing tool with complementary properties and functionality to Cas9 (Zetsche et al., 2017; Zhang et al., 2017).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Zetsche et al., 2017
The main-text passage comparing Cpf1 with Cas9 discusses smaller crRNA arrays and easier multiplex editing, not specificity or off-target effects. A search for those claims and for Cas13 found no passage in our copy that addresses the attributed comparison. (LLM, conf=0.76)
Experimental — accuracy not yet measuredTAP-seq is useful for functional genomics applications where phenotypes appear as expression changes in small gene sets.
“TAP-seq is applicable to a broad range of functional genomics applications, including studies of gene regulatory networks7,19,31, signaling pathways6,32, and combinatorial regulatory logic7,32,33, where phenotypes of interest are represented by expression changes in small sets of genes.”
From Targeted Perturb-seq enables genome-scale genetic screens in single cells (2020) · cites this paper as [33]
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- Authors
- Zetsche, B., Heidenreich, M., Mohanraju, P., Fedorova, I., Kneppers, J., DeGennaro, E. M., Winblad, N., Choudhury, S. R., Abudayyeh, O. O., Gootenberg, J. S., Wu, W. Y., Scott, D. A., Severinov, K., van der Oost, J., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Wageningen University & Research (NL)
- Rutgers, The State University of New Jersey (US)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2016-12-05
- Volume
- 35
- Issue
- 1
- Pages
- 31-34
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC5225075
- OpenAlex
- W2560230043
- Cited by
- 926
- References
- 15
- Citations by year
- 2026: 44 · 2025: 67 · 2024: 91 · 2023: 103 · 2022: 107 · 2021: 121 · 2020: 108 · 2019: 108 · 2018: 93 · 2017: 83 · 2016: 1
- FWCI
- 49.39
- Type
- journal article
- Language
- en
- Update notices
- erratum (2017-02-01)
- Open access
- bronze
- doi.org
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Socio-Environmental Systems Modeling (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- PMC
- Funders
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (714.015.001)
- Russian Science Foundation (14‐14‐00988)
- Skolkovo Institute of Science and Technology
- National Institutes of Health (GM10407, 5T32EB1680, 2t32eb001680-21)
- McGovern Institute for Brain Research, Massachusetts Institute of Technology
- National Institute of Mental Health (DP1MH100706, 5DP1-MH100706, R01MH110049)
- National Institute of General Medical Sciences (R01GM104071)
- National Institute of Biomedical Imaging and Bioengineering (T32-EB001680, 5T32EB1680)
- 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
- multiplex genome editing, CRISPR/Cas12a, mammalian cells, mouse brain
- MeSH terms
- Gene Editing, Animals, Bacterial Proteins, RNA, Bacterial, Sequence Analysis, RNA, Mice, CRISPR-Cas Systems, CRISPR-Associated Proteins
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