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

Autor: Jedrzejczyk DJ; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark., Poulsen LD; Artisan Bio, 363 Centennial Parkway, Suite 310, Louisville, CO, 80027, USA., Mohr M; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark., Damas ND; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark., Schoffelen S; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark., Barghetti A; Artisan Bio, 363 Centennial Parkway, Suite 310, Louisville, CO, 80027, USA., Baumgartner R; Artisan Bio, 363 Centennial Parkway, Suite 310, Louisville, CO, 80027, USA., Weinert BT; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark., Warnecke T; Artisan Bio, 363 Centennial Parkway, Suite 310, Louisville, CO, 80027, USA. tanya@artisancells.com., Gill RT; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800, Kongens Lyngby, Denmark. rtg@artisancells.com.; Artisan Bio, 363 Centennial Parkway, Suite 310, Louisville, CO, 80027, USA. rtg@artisancells.com.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2022 Jul 16; Vol. 12 (1), pp. 12193. Date of Electronic Publication: 2022 Jul 16.
DOI: 10.1038/s41598-022-15388-z
Abstrakt: 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.
(© 2022. The Author(s).)
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje