Structural And Functional Studies Of The ICP1 Phage Encoding CRISPR-Cas System Complex

The constant threat of phage predation leadsto the evolution of multiple immune mechanisms,which in turn leadsto the evolution of phage avoidance strategies,maintaining a delicate balance between the two.Among the immune mechanisms of bacteria against phage,CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins)system can specifically recognize foreign nucleic acid sequences and protect against invading phage.However,the existence of CRISPR-Cas system was also found in ICP1(The International Centre for Diarrhoeal Disease Research,Bangladesh cholera phage 1)phage in 2013.According to the genetic structure,it belongs to type I-F CRISPR-Cas system,which effectively targets the PLE(PICI-like element)island of Vibrio cholerae,destroying its phage resistance.So that the ICP1 phage can successfully "reproduce" offspring.In order to explore the similarities and differences between the CRISPR-Cas system of ICP1 phage and bacteria,we studied the structure and function of the CRISPR-Cas system of ICP1 phage.In terms of protein composition,the CRISPR-Cas system of ICP1 phage is the same as the type I-F1 CRISPR-Cas system of Pseudomonas aeruginosa,including six proteins: Cas1,Csa2/3,Csy1,Csy2,Csy3 and Csy4.Firstly,the structures of Csy3,Csy1-Csy2 and Csy complex(assembled by Csy1-Csy4 and crRNA)have been resolved by X-ray crystallography.The structural comparison between ICP1 Csy complex and Pseudomonas aeruginosa Csy complex showed obvious difference.The structure of Csy-dsDNA was resolved by cryo-electron microscopy.This study also found that Cas1 could bind Csy complex,which is different from the I-F system of bacteria.Later,according to the breakthrough point of Cas1 binding Csy complex,this study successfully prepared Csy-dsDNA-Cas1-Cas2/3 complex,and its structure was resolved by cryo-electron microscopy.The structure of Csy-dsDNA-Cas1-Cas2/3 contains two Csy-dsDNA complexes,which surround the heterohexamer of Cas1-Cas2/3 in the middle,forming a center symmetric structure.Based on the analysis of each binding interface,we hypothesized that N148/Q150/N151 of Csy1 might be involved in the recognition of PAM sequence to avoid the Csy complex targeting phage’s own DNA.The Csy2 subunit of the Csy complex bindsto Cas2/3,and the untargeted strand of the target DNA(the strand that does not complement and pair with crRNA)enters the Rec A domain of Cas2/3 from the surface of Csy1-Csy2.Cas2/3 then begins to degrade the untargeted strand DNA.The linker between Cas2/3 and Csy complex is a Cas1 dimer.One Cas1 bindsto Cas2/3,and one bindsto the Csy4 and Csy3 subunits of the Csy complex,which is different from the results of the current bacterial CRISPR-Cas system studies.Through the activity assay in E.coli,the importance of the amino acid residues at the binding interface in the above structure for the in vivo activity of the ICP1 CRSIPR-Cas system was verified,indicating that ICP1 Cas1 is essential for the Cas2/3 recruitment by ICP1 Csy-dsDNA.In conclusion,this study determined 3 crystal structures and 2cryo-electron microscopy structures by means of molecular biology,biochemistry and structural biology,and discovered a new way of recruiting Cas2/3 through Cas1 in the ICP1 phage CRISPR-Cas system.Furthermore,the in vivo activity assay confirmed the accuracy of the structure.Our results enrich the mechanism of Cas2/3 recruitment by the Csy-dsDNA complex of the I-F CRISPR-Cas system,and provide a reliable structural basis for the development of the ICP1 CRISPR-Cas system as a gene editing tool.