Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a.
Nucleic Acids Res
; 48(6): 3228-3243, 2020 04 06.
Article
em En
| MEDLINE
| ID: mdl-31989168
ABSTRACT
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. 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.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Reparo do DNA
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Endodesoxirribonucleases
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Proteínas Associadas a CRISPR
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Sistemas CRISPR-Cas
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Edição de Genes
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article