Nucleic acids research
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Nucleic acids research · Apr 2020
Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a.
Genome 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. ⋯ 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.