Interfering With Retrotransposition By Two Types Of CRISPR Effectors: Cas12a And Cas13a

CRISPRs are a promising tool being explored in combating exogenous retroviral pathogens and in disabling endogenous retroviruses for organ transplantation.The CRISPR-Cas systems(clustered regularly interspaced short palindromic repeat-CR ISPR-associated systems)are the immunity mechanism against invading foreign genetic material,which originated and evolved in bacteria and archaea.This system recognizes and destroys specific foreign sequences by forming cr RNA(CRISPR RNA)-effector protein complexes,such as phage virus and foreign DNA,and in some cases RNA.Based on the characteristics of the system,a series of efficient gene-editing tools have been developed,which are widely used to edit the genome of the hosts.The recently emerged class 2 type V CRISPR-Cas12 a protein,lacks the HNH domain,contains a Ruv C nuclease domain and a putative Nuc domain,which is responsible for the cleavage of two DNA strands,respectively.The Cas12 a has both DNA and RNA nuclease activities and is able to edit multiple genes simultaneously.Cas12 a can be used regulating heterologous DNA editing in bacteria,plants,and mammalian cells.The class 2 type VI CRISPR-Cas13 group of effectors were discovered and found to possess the unique ability to specifically target and cleave single-stranded RNA rather than double-stranded DNA substrates.Cas13 a contains two higher eukaryotes and prokaryotic nucleotide junction(HEPN)domains.Cas13 a is guided by a single cr RNA and can be programmed to cleave ss RNA targets carrying complementary protospacers,while the Cas13a-cr RNA complex is activated upon binding to target RNA and subsequently cleaves non-specifically ss RNA in host cells,resulting in the observed growth rate inhibition.Particularly,the latest study revealed activated Cas13 a provided bacterial hosts with a ‘passive protection' mechanism to defense against DNA phage infection by inducing cell growth arrest in infected cells,which is especially significant as it lets Cas13 a mount defense against both RNA and DNA invaders.In general,the Cas12 a and Cas13 a systems offer novel mechanisms of C RISPR actions and allow genetic manipulation across the central dogma,that has not been evaluated for retrovirus interference.Retrotransposon is a transposon element mediated by RNA,which synthesizes m RNA by transcription,and then synthesizes new DNA elements by reverse transcription to integrate into the genome to complete transposition.Tf1 is a retrotransposon from Schizosaccharomyces pombe,which encodes a long polyprotein,flanked by 385 base long terminal repeats(LTRs).LTR retrotransposons and retroviruses share many common features regarding their genome structure,replication mechanism,and life cycle.Both LTR retrotransposons and retroviruses replicate via reverse transcription and propagate by integrating into the host's genome,then finish copying and jumping in eukaryotic cells.Therefore,LTR retrotransposons can be used as an effective model for studying retroviruses.In this study,we first developed the CRISPR-C as12 a gene-editing tool in fission yeast strains,constructed a gene-editing system that targets yeast endogenous genes,and tested the editing efficiency of Cas12 a proteins in different subspecies.A Cas12 a protein with high editing efficiency in fission yeast cells was screened for Fn Cas12 a effector protein derived from the Francisella tularensis subsp.novicida U112 for subsequent experiments.Then,by fitting the LTR retrotransposon Tf1 in fission yeast as a model system,we repurposed CRISPR-Cas12 a and Cas13 a to interfere with Tf1 retrotransposition and evaluated their different mechanisms of action.Cas12 a targets the DNA stage of the Tf1 transposition process and exhibited strong inhibition on retrotransposition,allowing marginal Tf1 transposition that was likely the result of a lasting pool of Tf1 RNA/c DNA intermediates protected within virus-like particles(V LPs).Therefore,we further constructed new constructs for persistent cr RNA targeting and used Fn Cas12 a to interfere with the retroposition,as a result,the residual transposition activities of Tf1 were completely eliminated.On the other hand,we found that targeting CRISPR-Cas13 a to Tf1 RNA intermediates can also significantly inhibit Tf1 transposition.But unlike in bacterial hosts,the sustained activation of Cas13 a by Tf1 transcripts did not cause cell growth arrest in S.pombe,indicating virus-activating Cas13 a likely acted differently in eukaryotic cells.In summary,the study gained insight into the actions of novel CRISPR mechanisms in combating retroviral pathogens and established system parameters for developing new strategies in the treatment of retrovirus related diseases.