UMMS Affiliation

Department of Molecular, Cell and Cancer Biology; Department of Biochemistry and Molecular Pharmacology; Program in Molecular Medicine; Li Weibo Institute for Rare Diseases Research

Publication Date

2019-05-07

Document Type

Article

Disciplines

Biochemistry, Biophysics, and Structural Biology | Cell and Developmental Biology | Embryonic Structures | Genetic Phenomena | Genetics and Genomics | Nucleic Acids, Nucleotides, and Nucleosides

Abstract

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.

Rights and Permissions

Copyright The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

DOI of Published Version

10.1093/nar/gkz184

Source

Nucleic Acids Res. 2019 May 7; 47(8): 4169–4180. Published online 2019 Mar 20. doi: 10.1093/nar/gkz184. Link to article on publisher's site

Journal/Book/Conference Title

Nucleic acids research

Related Resources

Link to Article in PubMed

PubMed ID

30892626

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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