Optimization Of Cpf1 Base Editor And Its Application In Gene Editing Rabbits

Base editor(BE)is a breakthrough gene editing technique,which is widely used in the construction of single gene disease models and the correction of pathogenic site mutations.Cpf1 protein can recognize the protospacer adjacent motif(PAM)of T-rich primordial sequence,while Cas9 recognition of G-rich PAM,Cpf1 broadens the targeted genomic range.Ornithine transcarbamylase(OTC)deficiency is an X-linked disease,which accounts for nearly half of the urea cycle-related genetic diseases.OTC is one of the common enzymes in the urea cycle pathway.If the gene is mutated,ammonia will accumulate in the blood and damage the nervous system and liver.After the mutation of OTC gene in rabbits,the blood ammonia level will increase,and the liver will show a fibrotic phenotype,which can simulate the symptoms of human disease.It has been reported that the average editing efficiency of dLb Cpf1-BEs in endogenous sites is only 22%,which is lower than that of traditional n Cas9-BEs(the editing efficiency of BE4max is between 69 and 77%),which seriously hinders the wide application of dLb Cpf1-BEs.Therefore,the base editing efficiency of dLb Cpf1-BEs is improved by two optimization methods,and then the most efficient d Cpf1-e CDA1 is selected to verify in rabbit embryos and construct OTC gene editing rabbits.1)Therefore,in order to improve the editing efficiency of dLb Cpf1-BEs,we optimized it through three modification methods:crRNA^{t RNA},cr-HDV and U-rich crRNA,and verified the efficiency at six different sites.The results showed that the editing efficiency of U-rich crRNA at all sites was slightly improved(1.05 to 1.69times);cr-HDV only increased the editing efficiency of FANCF sites by 1.85 times,but decreased in two of the other five loci;for crRNA^{t RNA},only CDKN2A loci showed a slight improvement in editing efficiency.The above results show that the optimizing the 3-terminal sequence of crRNAs cannot significantly improve the base editing efficiency of d Cpf1-BE3.2)We further speculate that the low activity of rat cytosine deaminase(r A1)used in dLb Cpf1-BE3 is the main reason that affects editing efficiency,so we choose three more active deaminases(evo APOBEC1,evo CDA1 and A3A)to replace r A1 in dLb Cpf1-BE3,and construct three BEs systems:d Cpf1-e A1,d Cpf1-e CDA1 and d Cpf1-A3A.Base editing efficiency of d Cpf1-e A1,d Cpf1-e CDA1,d Cpf1-A3A and dLb Cpf1-BE3 were tested at the HEK293T cell level.Compared with dLb Cpf1-BE3,d Cpf1-e CDA1 significantly improved the base editing efficiency of 66.7%of the sites.And d Cpf1-e A1 and d Cpf1-A3A significantly improved the editing efficiency of 50%of the sites.Among them,when editing the sequence rich in GC,d Cpf1-e CDA1 can significantly improve the efficiency of C-T(3.17±0.80%VS 17.38±2.69%),while d Cpf1-A3A is not significant.In a word,the editing efficiency of d Cpf1-e CDA1 system was improved most significantly.3)The dLbCpf1-eCDA1 and dLb Cpf1-BE3 systems were compared at six sites of rabbit embryos.In TYR-1 site,the editing efficiency of dLbCpf1-eCDA1 was 11.75%and the editing efficiency of dLb Cpf1-BE3 was 3.67%.Except for TYR-1 site,the base editing efficiency of dLbCpf1-eCDA1 at the other five sites is more than 30%,with a maximum of 77.67%.In short,the editing efficiency of dLbCpf1-eCDA1 is between11.75-77.67%,while that of dLb Cpf1-BE3 is between 0-21.7%.It is revealed that dLbCpf1-eCDA1 can significantly improve the efficiency of base editing at the embryonic level.4)We applied dLbCpf1-eCDA1 to rabbit gene editing and introduced an early termination codon by targeting the fifth exon of OTC gene.Its editing efficiency was between 3.7%and 61.3%.It was further confirmed by RT-q PCR and western blotting that the expression of OTC in gene editing rabbits was significantly decreased.This study developed a dLbCpf1-eCDA1 system with high editing efficiency by optimizing the replacement of deaminase,and applied this system to construct rabbits with OTC gene mutation.