Structure Study Of The Complex Formed By NmeCas9 And Its Anti CRISPR Protein-Acr?C1_Boe

Based on the property that CRISPR/Cas system can cleavage foreign nucleic acids under the guidance of CRISPR-derived RNA(crRNA),CRISPR/Cas technology has been applied as an efficient gene editing tool in the research of tumors,infections and genetic diseases.Although this technique has been widely used,it still has some defects,such as the uncontrollable gene editing activity and off-target effect in vivo.Recent studies have found that phage-encoded anti-CRISPR proteins(Acrs)can inhibit gene editing activity of CRISPR/Cas system through various strategies.Besides Acrs can also be used to improve off-target effect of CRISPR/Cas system.Among all types of CRISPR/Cas systems,the gene editing activity of type ?-C system which has extremely low off-target effect als cannot be control.At present,Acrs inhibiting the gene editing activity of ?-C system have been successively reported.Clarifying the mechanism of these Acrs can provide theoretical guidance for the development of new tools for the regulation of gene editing activity of type ?-C type systems.Among these Acrs,Acr?C1Boe encoding by Brackiella oedipodis phages has not been clarified so far.Alhough the mechanism of the Acr?C1Nme,a homologous protein derived from Neisseria meningitides phages,has been elucidated,evolutionary analysis and mechanism comparison of Acrs revealed that the mechanism of homologous Acrs also have different.Therefore,Acr?C1Boe may also have unique functions,and it still needs in-depth study.In this paper,we determined the high-resolution crystal structure of the Acr?C1Boe(2.2A)and the Acr?C1Boe-HNHNme complex(1.9 A)by structural biology methods.Through analysing the structure of Acr?C1Boe-HNHNme complex,and comparising its interaction with the Acr?C1Nme-HNHNme and the activated NmeCas9 ternary complex structure,we found that the Acr?C1Boe can interfering crRNA loading and affecting nuclease activity.In addition,the key binding and active sites of Acr?C1Boe were analyzed through biochemical experiments.These results can provide a theoretical basis for the development of regulatory tools for ?-C CRISPR/Cas system.