Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array
Open accessAbstract
Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.
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Several methods have been described to limit continuous expression of lentiviral transgenes, such as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits.
“Several methods have been described to limit continuous expression of lentiviral transgenes as natural promoter silencing and self-limiting CRISPR-Cas genetic circuits41,59,60.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [59]
The paper’s lentiviral-vector discussion (page unavailable) describes Cpf1 cleavage of viral RNA during vector production and a design change that permits functional virions. It does not describe natural promoter silencing or a self-limiting CRISPR-Cas circuit that limits continuous transgene expression. Searches for those mechanisms found no supporting passage in our copy. (LLM, conf=0.75)
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References 19 · Cited by 9
Research works printed but not linked: 2
- Ostlund, C., Folker, E.S., Choi, J.C., Gomes, E.R., Gundersen, G.G., and Worman, H.J. (2009). Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins. Journal of cell science 122, 4099-4108.
- Xie, K., Minkenberg, B., and Yang, Y. (2015). Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proceedings of the National Academy of Sciences 112, 3570-3575.
- 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)
- ORCID
- Venue
- bioRxiv (Cold Spring Harbor Laboratory)
- Publisher
- openRxiv
- Published
- 2016-10-01
- OpenAlex
- W2949839430
- Cited by
- 9
- References
- 19
- Citations by year
- 2019: 4 · 2018: 2 · 2017: 2 · 2016: 1
- Type
- posted content
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- Funders
- McGovern Institute for Brain Research, Massachusetts Institute of Technology
- 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, multiplex genome editing, RNA-guided genome editing
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