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

Author:

Maltseva Ekaterina A.ORCID,Vasil’eva Inna A.ORCID,Moor Nina A.ORCID,Kim Daria V.ORCID,Dyrkheeva Nadezhda S.ORCID,Kutuzov Mikhail M.ORCID,Vokhtantsev Ivan P.ORCID,Kulishova Lilya M.ORCID,Zharkov Dmitry O.ORCID,Lavrik Olga I.ORCID

Abstract

AbstractCRISPR/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 betweenStreptococcus pyogenesCas9 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.

Publisher

Cold Spring Harbor Laboratory

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