CRISPR-Cas12a nucleases function with structurally engineered crRNAs: SynThetic trAcrRNA

Jedrzejczyk, D. J.; Poulsen, L. D.; Mohr, M.; Damas, N. D.; Schoffelen, S.; Barghetti, A.; Baumgartner, R.; Weinert, B. T.; Warnecke, T.; Gill, R. T.

doi:10.1038/s41598-022-15388-z

Title: CRISPR-Cas12a nucleases function with structurally engineered crRNAs: SynThetic trAcrRNA

Journal Article · 15 July 2022 · Scientific Reports

DOI: https://doi.org/10.1038/s41598-022-15388-z · OSTI ID:1876454

Jedrzejczyk, D. J.; Poulsen, L. D.; Mohr, M.; Damas, N. D.; Schoffelen, S.; Barghetti, A.; Baumgartner, R.; Weinert, B. T.; Warnecke, T.; Gill, R. T.

Abstract CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate sixteen types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo . We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region led to comparable genome editing efficiencies. In conclusion, we show that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications.

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Research Organization:: Univ. of Colorado, Boulder, CO (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC)

Grant/Contract Number:: SC0018368

OSTI ID:: 1876454

Journal Information:: Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 12; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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