TY - JOUR

T1 - Key thermodynamic characteristics of Cas9 and Cas12a endonucleases’ cleavage of a DNA substrate containing a nucleotide mismatch in the region complementary to RNA

AU - Baranova, Svetlana V.

AU - Zhdanova, Polina V.

AU - Pestryakov, Pavel E.

AU - Chernonosov, Alexander A.

AU - Koval, Vladimir V.

N1 - This research was partially supported by the Russian Science Foundation (grant No. 20-14-00214) and by the Russian state-funded project for ICBFM SB RAS (grant No. FWGN-2025-0020).

PY - 2025/7/1

Y1 - 2025/7/1

N2 - CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates—containing a mismatch in the region complementary to RNA—by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a–crRNA complex and compared it with Cas9–sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a–crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9–sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.

AB - CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates—containing a mismatch in the region complementary to RNA—by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a–crRNA complex and compared it with Cas9–sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a–crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9–sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.

KW - CRISPR/Cas

KW - Cas12a activity

KW - Cleavage

KW - Molecular dynamics simulation

KW - Oligonucleotide mismatch

KW - Thermodynamics

UR - https://www.mendeley.com/catalogue/bef6ab8c-7b53-394b-93be-19eb55d6084d/

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-105004290037&origin=inward&txGid=affc5fab168b4244cd4906abcbb7fa5a

U2 - 10.1016/j.bbrc.2025.151892

DO - 10.1016/j.bbrc.2025.151892

M3 - Article

C2 - 40334424

VL - 768

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

M1 - 151892

ER -