The Safety Evaluation And Technology Development Of CRISPR/Cas13 System

1.The safety evaluation of CRISPR/Cas13 systemCRISPR/Cas13 system is a programmable RNA-guided RNA-targeting system consisting of Cas13 and CRISPR RNA(cr RNA).Cas13 possesses two distinct RNase activities,one is responsible for cr RNA maturation,and the other is activated when the Cas13-cr RNA complex binds to the target RNA,which not only cleaves the target RNA in cis but also promiscuously cleaves bystander RNAs in trans.This trans-cleavage activity triggered by the binding of the target RNA is referred to as collateral activity.Even though the collateral activity of Cas13 was obviously observed in bacteria and in vitro,it was initially not detected in mammalian cells.Therefore,CRISPR/Cas13 system has been widely applied to transcriptome engineering in mammalian cells with potentially important clinical applications.However,it is still controversial whether Cas13 exhibits collateral activity in mammalian cells.Here,we accidentally found that knockdown of gene expression using Rfx Cas13 d in the adult mouse brain neurons caused the death of mice.The death would occur when target genes were present and significantly knocked down,but it was not due to the loss of target gene function or off-target effects,which suggested that the collateral activity of Rfx Cas13 d might be the cause of death.Mechanistically,we showed that Rfx Cas13 d exhibited collateral activity in mammalian cells,which was positively correlated with the abundance of the target RNA.The collateral activity of Rfx Cas13 d cleaved 28 s r RNA into two fragments,leading to translation attenuation and activation of ZAKα-JNK/p38-immediate early gene(IEG)pathway.In conclusion,our findings warned that the application of CRISPR/Cas13 system in mammalian cells is not as safe as we thought.It is necessary to re-evaluate carefully the safety of CRISPR/Cas13 system and re-examine the experimental conclusions obtained by using CRISPR/Cas13 system before.Moreover,our exploration of the mechanism of the collateral activity of Rfx Cas13 d,on the one hand,can guide the future optimization of Cas13,making it a more perfect RNA knockdown technology by reducing or eliminating collateral activity;on the other hand,it can also inspire us to develop new applications taking advantage of Cas13’s collateral activity in mammalian cells.2.The technology development based on CRISPR/Cas13 systemRNA and protein are interconnected biomolecules that can influence each other’s life cycles and functions through physical interactions.Abnormal RNA-protein interactions could lead to cellular dysfunction and human diseases.Therefore,mapping networks of RNAprotein interactions is crucial for understanding cellular processes and the pathogenesis of related diseases.Currently,various practical protein-centric methods for investigating RNAprotein interactions have been reported,but few robust RNA-centric methods exist.Collateral activity is a huge safety concern for Cas13,but not for catalytically dead Cas13(d Cas13).d Cas13 is not able to cleave RNA but retains its programmable RNA-binding activity.We constructed a d Cas13-PBL(proximity labeling enzyme)fusion protein,which can specifically bind to the target RNA under the guidance of the cr RNA and label its interacting proteins.These labeled proteins were easily enriched and identified.We named this method CRISPR-based RNA proximity proteomics(CBRPP).After several optimizations,we successfully identified the RNA-binding proteins(RBPs)of ACTB m RNA and long noncoding RNA(lnc RNA)NORAD using CBRPP.In summary,CBRPP is a new RNA-centric in vivo method to study RNA-protein interactions.Compared with previous methods,this method is simple and fast,and does not require cross-linking,in vitro manipulation of RNA-protein complexes,or pre-editing of target RNAs.CBRPP is a powerful complement to existing methods,providing researchers with a new RNA-centric method to investigate RNA-protein interactions.