Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems

Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems Full article

Journal

International Journal of Molecular Sciences
, E-ISSN: 1422-0067

Output data

Year: 2022, Volume: 23, Number: 22, Article number : 13889, Pages count : DOI: 10.3390/ijms232213889

Tags

Cas9 activity; cleavage; CRISPR-Cas system; oligonucleotide mismatch; thermodynamics

Authors

Baranova S.V. ¹ , Zhdanova P.V. ¹ , Lomzov A.A. ^1,2 , Koval V.V. ^1,2 , Chernonosov A.A. ¹

Affiliations

1	Institute of Chemical Biology and Fundamental Medicine SB RAS
2	Novosibirsk State University

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson–Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the “seed” region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the “unwinding” of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.

Baranova S.V. , Zhdanova P.V. , Lomzov A.A. , Koval V.V. , Chernonosov A.A.
Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems
International Journal of Molecular Sciences. 2022. V.23. N22. 13889 . DOI: 10.3390/ijms232213889 WOS Scopus РИНЦ OpenAlex

Published print:

Nov 9, 2022

Web of science:	WOS:000887442100001
Scopus:	2-s2.0-85142624019
Elibrary:	57633464
OpenAlex:	W4308973558

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