Generation Of Rabbit Models With CRISPR-STOP And CRISPR Start-Loss

CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins)technolohy has been widely used to characterize gene functions in the past few years because of its simplicity and efficacy.CRISPR/Cas is RNA-directed adaptive immune system which originally derives from bacteria and archaea.There are two components in CRISPR/Cas system: Cas enzymes and a single guide RNA(sg RNA).The technology has been used in early days to identify gene function by means of gene kock-out and CRISPR interference.However,these methods are not perfect:(1)KO generates double strand breaks at terget sites,the repairment of which results in indels(insertions and deletions).(2)DSBs have been reported to trigger ontarget m RNA misregulation and complicated genomic rearrangements.CRISPRi is mediated by catalytically dead Cas9(d Cas9).(3)In the case of CRISPRi,the sg RNA directs d Cas9 binding to specific DNA sequences,which could block transcription of target genes.This method can only manipulate gene expression temporarily,and does not thoroughly inhibit gene expression.Base editors,a combination of CRISPR/Cas9 nickase and deaminases,install C.G to T.A conversion in genome,resulting in the conversion of CAA,CAG,CGA or TGG into stop codons(TAA,TAG,TGA)in a target gene.As a result,the target genes are silenced.This method is referred to as CRISPR-STOP.It disrupts gene expression without generating DSBs.It can mimic 16.2% of pathogenic mutations and 32,000 nonsense mutations related to cancer.Therefore,CRISPR-STOP can be a powerful tool to generate animal models for the study of treatment for many diseases.In this study,we first tested BE3-mediated CRISPR-STOP strategy in rabbit embryos and then introduced a premature stop codon in the Mstn gene of rabbit using the strategy.We demonstrated that the editng efficiency in rabbit embryos was 75%-87%,and founder rabbits showed an editing efficiency of up to 96%.The fact that BE3 recognizes NGG PAM and that editable codons are limited restricts the targetable range in the genome to a large extent,which makes many cancer-related nonsense mutations impossible to mimic.To solve this problem,we constructed NG-BE4 max,n Nme2-BE4 max and Spymac-BE4 max which recognize NGN PAM,N4 CC PAM and NAAN PAM,respectively.We proved that those base editors efficiently mediated CRISPR-STOP strategy to introduce premature stop codon in rabbit embryos.NG-BE4max-mediated CRISPRSTOP achieved an editing efficieny of 75%-100% in rabbit embryos and 49%-97% in founder rabbits.These figures for n Nme2-BE4max-mediated CRISPR-STOP were 17.3%-52.5% and 20%-50%,respectively;For Spy-mac-BE4max-mediated CRISPR-STOP,the figures were 28.00 %-100.00 % and 100%,respectively.Even though CBEs-mediated CRISPR-STOP efficiently introduces premature stop codon in target genes,they were reported to trigger exon-skipping.To compensate for the limitation,we proposed a novel gene silencing method termed CRISPR StartLoss.It relies on the capability of both ABEs and CBEs to convert a start codon(ATG)into(GTG,ACG or ATA)non-start codons.CRISPR Start-Loss can be mediated by more stringent ABEs,attenuating potential off-target editing,and it targets 1074 genes which cannot be silenced by CRISPR-STOP.In this study,seven target sites in five genes(Tyr,Otc,Timeless,Lmna,Pah)were tested in the human embryonic kidney cell line 293T(HEK293T)as a proof of concept by co-transfecting BEs-coding and corresponding guide RNA plasmids.It presents an average efficiency between 11.63% and 30.67%.Subsequently,seven sg RNAs targeting different bases of start codon in four genes(Otc,Tyrp1,Hbb2,Fgf5)were selected to verify CRISPR Start-Loss strategy in rabbit embryos.BEs-encoding m RNAs and corresponding in vitro transcribed sg RNAs were microinjected into zygotes.After developing to blastula stage,they were collected and subjected to Sanger sequencing.We demonstrated that CRISPR Start-Loss exhibited an average editing efficiency ranging from 15.68% to 73.50 % in rabbit embryos.Encouraged by the results from cells and embryos,we applied CRISPR Start-Loss strategy to generate two animal models.A single G·C to A·T base pair conversion was designed in the start codon of Fgf5 intending to disrupt it.Comparison was made between body weight,hair length of wild type(WT)rabbits and engineered Fgf5^-/- rabbits.Together with Fgf5 m RNA expression and protein expression results,we illustrated that disruption of start codon led to substantial decrease in Fgf5 m RNA and protein.In addition,start codon destroyed Fgf5 rabbits showed significantly longer hair than WT rabbits.Similarly,start codon destroyed Otc rabbits showed lower Otc m RNA expression and protein expression.The overall survival rate decreased,and some of them showed fatal hyperammonemia,liver fibrosis.In summary,we demonstrated that CRISPR Start-Loss is a practical method to silence genes of interest.Taken together,we tested the feasibility of CBEs-mediated CRISPR-STOP strategy in rabbit embryos and constructed NG-BE4 max,n Nme2-BE4 max and Spymac-Be4 max to expand the targeting range to NG-rich,C-rich and A-rich sequences.In addition,we proposed a novel gene silencing strategy,CRISPR Start-Loss,which silences genes by disrupting start codon with base editors.We initially tested the feasibility of this strategy in HEK293 T cells and rabbit embryos.Fgf5 and Otc Start codon destroyed rabbits verified that CRSIPR Start-Loss strategy is an efficient and practical method to silence genes.It is complementary to CRISPR-STOP strategy.