• Sjoerd C A Creutzburg, Wen Y Wu, Prarthana Mohanraju, Thomas Swartjes, Ferhat Alkan, Jan Gorodkin, Raymond H J Staals, and John van der Oost.
• Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
• Nucleic Acids Res. 2020 Apr 6; 48 (6): 3228-3243.

AbstractGenome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

22 keywords...

hide…