TY - JOUR

T1 - Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

AU - Maltseva, Ekaterina A

AU - Vasil'eva, Inna A

AU - Moor, Nina A

AU - Kim, Daria V

AU - Dyrkheeva, Nadezhda S

AU - Kutuzov, Mikhail M

AU - Vokhtantsev, Ivan P

AU - Kulishova, Lilya M

AU - Zharkov, Dmitry O

AU - Lavrik, Olga I

N1 - This research was supported by Russian Science Foundation (grant 21-64-00017). Partial salary support from the Russian Ministry of Science and Higher Education (State funded budget project 121031300056-8 to D.O.Z.) is acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2023 Maltseva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2023/11/29

Y1 - 2023/11/29

N2 - CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

AB - CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

UR - https://www.mendeley.com/catalogue/673ad301-71bf-3731-a980-e4c3cd3d9cca/

U2 - 10.1371/journal.pone.0294683

DO - 10.1371/journal.pone.0294683

M3 - Article

C2 - 38019812

VL - 18

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 11

M1 - e0294683

ER -