Research On A Far-red Light-induced CRISPR/Cas9 Gene Editing System

CRISPR/Cas(Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)system has been developed to be an efficient gene-editing tool for genome modification of cells and organisms in recent years.The type II CRISPR/Cas9 system only consists of a Cas9 exonuclease and guide RNA(gRNA)to exert its biological function.CRISPR/Cas9 uses a gRNA as a guide to find the complementary protospacer DNA target in the specific genome where it cuts the double stranded DNA to achieve genome editing.In addition,its variants—catalytically inactivated Cas9(dead Cas9,dCas9)which is produced by inactivating the two catalytic domains(HNH and RuvC)of Cas9 can still tightly bind to target DNA sequence to directly control the target gene expression or regulate gene transcription by recruiting a variety of effector proteins.Light is ubiquitous and easy to obtain in nature.It is also an ideal inducer of gene expression because of highly tenability,non-toxicity and high spatiotemporal resolution.In this study,we designed and constructed a novel far-red light-induced CRISPR/Cas9(FCas9)gene editing system based on the photosensitive guanylic acid cyclase Bphs activated by far-infrared light(730 nm)irradiation,which enables accurate genome editing with high efficiency and specificity.The FCas9 system was further optimized from four aspects including spliting the Cas9 protein domain,different promoter,transcription activator and RNA binding protein,the system can be activated to edit the target gene under far-infrared light illumination in mammalian cells.In addition,the FCas9 system was characterized by a series of kinetic experiments including light time,light intensity,different cell lines and different genes.The results showed that FCas9 system has a positive correlation with light intensity and illumination time,and can be functional in different mammalian cell lines.At the same time,the far-red light-induced CRISPR/dCas9 system were also designed and constructed to successfully activate exogenous or endogenous target gene transcription by using the optimized system with good performance.The FdCas9 system was also characterized by illumination time,light intensity,different cell lines and spatiotemporal specificity.The results showed that the FdCas9 system was also dependent on the light intensity and light duration,and the system was also high spatiotemporal specificity and could be used in different mammalian cell lines.Finally,the FdCas9 system was applied to epigenome editing by combining the TET1(Ten-eleven translocation 1 protein)protease with the FdCas9 system,and then a variety of endogenous multiple methylated genes transcription was successfully activated.In summary,we have developed a new far-red light-controlled CRISPR/Cas9 gene editing system to rapidly modulate the genome editing with high efficiency and specificity.This system will be regarded as a powerful tool for genome engineering in in biomedical research and open up a new avenue for inducible CRISPR/Cas9 gene editing system.