Molecular Mechanism Of The Type I-F CRISPR-Cas System Inhibition By The Anti-CRISPR Protein AcrF3

CRISPR-Cas adaptive immune system originated from bacteria and archaea to combat invaders such as viruses and plasmids,which involves three steps:spacer adaptation,crRNA biogenesis and DNA interference.During DNA interference,the effector enzymes of CRISPR-Cas systems,such as Cas9 and Cas3,bind at specific sites on foreign genomes under the guidance of crRNA,then a single or double DNA break can be generated by the nuclease domain of these enzymes.This characteristic has been employed by researchers to obtain at precise target sites DNA breaks that can be repaired through intrinsic homology-directed repair(HDR),which requires a donor DNA template to fulfil the purpose of genome editing.Compared with other genome editing tools,CRISPR-Cas system has great advantages in that its guidance crRNA is readily engineered and it can cleave multiple distinct target sequences in parallel,making it one of the leading-edge and most active research areas in the field of biology recently.CRISPR-Cas system is able to fundamentally cure many kinds of genetic diseases such as sicklemia and can help researchers to effectively obtain gene knockout animals or cell lines.It may have exciting and widespread applications in clinic therapy and biomedical research.However,Cas9,Cas3 and other effector enzymes of CRISPR-Cas systems bear off-target problems,which will cause unpredictable genome editing and result in serious safety problems in biotherapy and other applications.How to inhibit these CRISPR-Cas effctors effectively and selectively in temporal and spatial dimensions has been an important scientific question.During coevolution with bacteria,the bacteriophages have also evolved unique tools,namely anti-CRISPR proteins,to inhibit the CRISPR-Cas systems.It was reported that a novel anti-CRISPR phage protein AcrF3 could effectively inhibit the type I-F CRISPR-Cas system effector enzyme Cas3.However,the molecular mechanism remains elusive.It will be of significance for CRISPR-Cas system effctor Cas3 activity regulation to uncover the molecular mechanism of the anti-CRISPR protein AcrF3 function.We firstly expressed and purified Pseudomonas aeruginosa effector Cas3(PaCas3)and AcrF3.It was found that AcrF3 functions as a homodimer to interact with PaCas3.PaCas3 has the divalent metal ion-dependent nuclease activity to cleave ssDNAs.We further determined the PaCas3-AcrF3 complex structure at 2.6 A resolution and found that PaCas3 harbors a separated N-terminal Cas2 domain,which enables PaCas3 to be engaged in both spacer adaptation and DNA interference.AcrF3 dimerizes in an inversion-symmetrical manner and then binds the concave surface of PaCas3 between the long linker region and CTD via extensive interactions with the HD domain,the linker region,RecA2,and the CTD.We conclude from analysis of all the aspects of AcrF3 function,that AcrF3 homodimer locks PaCas3 in an ADP-bound inactive form,prohibits PaCas3 from accessing the substrate DNA and its recognition by the upstream Csel subunit of Cascade,which leads to the inhibition of the type I-F CRISPR-Cas system.In addition,biochemical assays showed that AcrF3 binds the type I-F PaCas3 rather than the tested Cas3 from the type I-E CRISPR-Cas system.Sequence alignment and interaction analysis revealed that,except for Gln202,all 23 interacting residues in PaCas3 are distinct from those of type I-E Cas3s,hence these residues may determine the specificity of AcrF3 for PaCas3.The PaCas3-AcrF3 complex structure reported by us is the first structure of a CRISPR enzyme with an anti-CRISPR protein.It is the first time to reveal one of the inhibition mechanisms of CRISPR-Cas systems by anti-CRISPR proteins.This study also revealed the novel biochemical mechanism and structural characteristics of type I-F CRISPR-Cas system effector enzyme.It not only depicts a wonderful snapshot of the bacteria-phage coevolutionary arms race,but also sheds light on safe applications of the CRISPR-Cas genome editing systems.