Molecular Mechanism Of Staphylococcus Aureus Cas9 Inhibited By Acriia14 Protein

In response to phage invasion,bacteria have evolved CRISPR-Cas systems.As the adaptive immune system of bacteria,CRISPR-Cas systems function including three stages: spacer acquisition,cr RNA generation and DNA interference.According to the number of effector molecules in the interference phase,it can be divided into two categories: class I and class Ⅱ.The interference phase in the class I system requires the participation of multiple effector proteins,which are subdivided into types I,ⅡI and IV.In the class Ⅱ system,there are only single effector protein,including types Ⅱ,V,and VI systems.Given the ability of CRISPR-Cas systems to specifically cleave double-stranded DNA,they have been gradually developed into an efficient gene editing tool.Among them,Streptococcus pyogenes Cas9(Spy Cas9)in the type Ⅱ CRISPR-Cas system is the most widely used.However,because of its large molecular weight(150 k Da),it is not easy to transfect cells thus affecting its application.Therefore,many researchers are currently looking for more pocket Cas proteins that can replace Spy Cas9.Staphylococcus aureus Cas9(SauCas9)which also belongs to the type Ⅱ CRISPR-Cas system,is one of the good candidates,approximately 300 amino acids less than Spy Cas9,and also has high intracellular editing efficiency as well as low off-target effect.Although CRISPR-Cas system is a very powerful gene editing tool,there are still many problems in practical applications,one of the most important problems is off-target effect,which brings many uncertain factors to its application.Therefore,it is particularly important to study the regulatory mechanisms of CRISPR-Cas systems.Studies have found that in the face of this evolutionary pressure on bacteria,phages have also evolved corresponding antagonistic mechanisms.The proteins that function are called Anti-CRISPR proteins(Acrs).As suppressor proteins of CRISPR-Cas systems,Acrs can act as a switch to regulate gene editing.AcrⅡA14 was found to inhibit the activity of the SauCas9 protein,but its specific molecular mechanism is unknown.Therefore,studying the inhibition mechanism of AcrⅡA14 helps us to better utilize the SauCas9 system,but also expands our understanding of the mechanism by which Acrs inhibits CRISPR-Cas systems.We first demonstrated that AcrⅡA14 protein exerts its inhibitory function by directly interacting with SauCas9 protein by in vitro cleavage assays,GST pulldown assays and gel exclusion chromatography assays.We also found that its binding does not affect the binding of SauCas9 protein to sgRNA and dsDNA.In addition,it was found that its C-terminal domain(AcrⅡA14ct)was mainly involved in the inhibitory function,and the N-terminal HTH domain was dispensable in the inhibitory process.Further,we interrogated the inhibitory activity of AcrⅡA14 against different Cas9 protein and found that it had no inhibitory effect on the Spy Cas9 and Nme Cas9 proteins.This illustrates that AcrⅡA14 is a specific inhibitor of SauCas9.To elucidate the molecular mechanism by which AcrⅡA14 specifically inhibits SauCas9 protein,AcrⅡA14 ct and SauCas9 proteins were first expressed and purified.Then,a stable SauCas9-sgRNA-AcrⅡA14ct-dsDNA quaternary complex protein was obtained by in vitro assembly.The structure of the SauCas9-sgRNA-AcrⅡA14ct-dsDNA complex was solved by X-ray crystallography at a resolution of 2.22 ?.The crystal structure shows that the AcrⅡA14 ct protein exists as a monomer consisting of four β-sheet sheets and four α helices located on one side.In the complex,SauCas9 adopts a bi-lobed structure,comprising recognition(REC)and nuclease(NUC)lobes.The channel formed by these two lobes binds the sgRNA-target DNA heteroduplex,as described previously.AcrⅡA14 ct binds to the SauCas9 complex with a 1:1 stoichiometry partially interacting with the L2 linker and making extensive contacts with the HNH domain which suggests that this interaction is responsible for SauCas9 inhibition.Close inspection of the interface between AcrⅡA14 ct and the HNH domain shows that β2,loop α2-β4,loop β4-α3 and C-terminus of AcrⅡA14 ct make extensive intermolecular hydrogen bond contacts with α4-α5 of the HNH domain and the loop between the HNH domain and the L2 linker.Based on the structural information,we designed the mutant assays and confirmed the correctness of the structure.Together,these structural and biochemical results demonstrate that AcrⅡA14 ct inhibits SauCas9 activity by directly binding to SauCas9 HNH domain and preventing its movement towards the cleavage site.Further,comparing the structure of the SauCas9-sgRNA-AcrⅡA14ct-dsDNA complex with the previously reported SauCas9-sgRNA-dsDNA complex,it was found that the binding of AcrⅡA14 ct to the HNH domain caused the proximity of the HNH domain to the nucleic acid hybrid strand,which also resulted in a significant allostery of the L1 linker.Further analysis revealed that the allostery of these two domains resulted in the formation of two new intramolecular interaction surfaces.Residues Arg617 on the HNH domain and Lys485,Lys489 on the L1 linker form interactions with the nucleic acid backbone of the target DNA strand and the sgRNA,respectively.Mutation of these three residues was confirmed to be able to reduce the ability of AcrⅡA14 ct to inhibit SauCas9.These data indicate that AcrⅡA14 ct is not only able to inhibit SauCas9 activation by directly binding to the HNH domain,thereby preventing its proximity to the substrate dsDNA.It can also further enhance its inhibitory activity by inducing allostery of the HNH domain and L1 linker of the SauCas9 protein.In previous reports it has been found that AcrⅡC1 and AcrⅡC3 proteins also inhibit the activity of Cas9 protein by directly binding to the HNH domain.By structural comparison,the binding region of AcrⅡA14 to the HNH domain was found to be significantly different from that of AcrⅡC1 and AcrⅡC3 proteins.Sequence conservation analysis shows that the residues bound by AcrⅡA14 are non-conserved,which also explains its specificity for SauCas9 inhibition.In addition,it is not found that the binding of AcrⅡC1 and AcrⅡC3 proteins to the HNH domain can also cause allosteric effects similar to AcrⅡA14 binding on Cas9 protein.In the present research paper,we solved the crystal structure of the SauCas9-sgRNA-AcrⅡA14ct-dsDNA quaternary complex at 2.22 ? resolution.The molecular mechanism by which AcrⅡA14 protein specifically inhibits SauCas9 activity was elucidated by structural analysis and biochemical assays.The mode of binding of AcrⅡA14 to SauCas9 and the means of multi-mechanism inhibition are different from previously reported Acrs.This finding expands our understanding of Acrs inhibiting CRISPR-Cas systems and provides a rationale for our better use of CRISPR-Cas systems,especially the SauCas9 system.