Structural Basis Of Cas12i1-crRNA Binary Complex

CRISPR-Cas system was first discovered in bacteria and archaea and provides resistance to phage infection.Bacteria and archaea that have the CRISPR system can "remember" information about invading foreign nucleic acids and provide adaptive immunity.After nearly three decades of research,scientists have differentiated the CRISPR-Cas system functionally and structurally.The CRISPR-Cas system,which consists of a Cascade complex of multiple Cas proteins to exert the function of targeted interference,is classified as the Class I.Such CRISPR-Cas system is limited in application due to the relatively complex genome and the multi-subunit composition of the effect complex.The Class II CRISPR-Cas system is composed of simpler components.It is composed of a single Cas protein containing multiple domains to play interference function.Such Cas proteins are usually more complex in structure,and multiple domains perform different functions respectively.Because of the simplicity of the Class II CRISPR-Cas system and its ability to reprogram cleavage sites,the most famous Type II CRISPR-Cas9 system,,has been used for genome editing in mammalian cells since 2013.After that,the newly discovered type-V CRISPR-Cas system has a different cleavage mode from Cas9,among which Cas12 a and Cas12 b have been successfully applied in genome editing.The study of the newly discovered CRISPR-Cas system will help expand the approach to genome editing and provide a deeper understanding of the evolutionary analysis of CRISPR.Cas12i1 is a newly discovered effector protein in Class II type-V CRISPR-Cas system.The activity of Cas12i1 in cutting Non-target strand(NTD strand)of ds DNA substrate is significantly higher than that in cutting Target strand(TD strand).This cleavage activity of Cas12i1 can be modified for highly specific genome editing.In this paper,the structure of Cas12i1-crRNA binary complex was analyzed in detail.Combined with biochemical experiments,the function and mechanism of Cas12i1 were summarized.Cas12i1 cleaves DNA substrates by two different mechanisms: Cis cleavage and Trans cleavage.The Cis cleavage activity of Cas12i1 refers to the sequence-specific directional cleaved of the target ds DNA complementarily paired with crRNA by Cas12i1 under the guidance of crRNA,which usually causes a Double strand break(DSB)in the target ds DNA.In the in vitro biochemical analysis experiments,we also observed that the cleavage activity of Cas12i1 on the Non-complementary strand(NTD)of the target ds DNA with crRNA was significantly higher than that of the complementary strand TD of the target double-stranded DNA.AndCas12i1 cleavage the non-specific ss DNA(Trans Cleavage)after the binding of target DNA and conformational change.Cas12i1 can bind any ss DNA in the environment,perform sequence nonspecific cleavage,and perform continuous cleavage starting from the5 'end of ss DNA.In addition,pseudo target oligonucleotides from Escherichia coli binding to crRNA were observed in the crystal structure of Cas12i1-crRNA binary complex obtained by us,which sim?lated the recognition of target DNA and crRNA.At the same time,after the superposition analysis with the Cas12i1-crRNA-target ternary complex analyzed by other members of our laboratory,it was found that the PI(PAM Interaction)domain in the binary complex underwent an open-off dynamic change process during the binding process of Cas12i1 to the target ds DNA.And other domains' conformational changes were observed.This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.