The Development Of Enhanced Prime Editor And Its Application In Sheep Fetal Fibroblasts

Superior breeds of livestock are essential to the development of animal husbandry.However,the conventional breeding is time-consuming and inefficient,which restricts the development of the livestock industry.The emergence of gene editing-based molecular design breeding has greatly accelerated the breeding program.The BMPR1 B gene is the first major gene identified to be associated to the high fecundity of sheep,where the g.A746 G mutation is significantly related to the litter size of sheep.The introgression of this mutation into sheep genome by gene editing could largely enhance the fecundity of sheep.To date,the prime editors(PEs)are the latest genome editing tools,and the canonical PE3 consists of three components: the effector,the prime editing guide RNA(peg RNA)and the nicking single guide RNA(sg RNA).The effector of PE is termed PE2,which is generated by fusing a reverse transcriptase(RT)with a Cas9 H840 A nickase;and the peg RNA consists of a sg RNA with a3′ extension containing the RT template(RTT)and primer binding site(PBS).With new DNA strand synthesized by reverse transcription,PE rewrites gene by replacing the original DNA strand.PE can install all 12 types of point mutations,small insertions and deletions,and can be applied to the genetic improvement of crops and livestock.However,PE was limited by several shortcomings which hinder its application: PE had not been demonstrated in mammalian embryos;and the editing efficiency of PE was low compared to other genome editing tools,requiring further improvement.Therefore,this study was conducted to generate gene-edited mice by PE3;and developed a new prime editor with higher efficiency termed enhanced prime editor(ePE);and the ePE was used for inducing g.A746 G mutation into the BMPR1 B gene in sheep fetal fibroblasts.The main results are as follows:1.The generation of Hoxd13 gene-edited mice by PE3To demonstrate the effectiveness of PE in mammalian embryos,the efficiencies of PE on human and mouse were evaluate at first,and the peg RNAs targeting three mouse loci(Hoxd13-1,Hoxd13-2 and Ar-2)were optimized on the lengths of RTT and PBS for higher efficiencies of 48.5%,21.4% and 13.2%,respectively.Subsequently,PE3 was injected into mouse zygotes in the form of RNAs,and the zygotes were then cultured into blastocysts followed by genome extraction for detection.The editing efficiencies of Hoxd13-1 and Hoxd13-2 loci were 44.4%(8/18)and 75.0%(12/16),much higher than that of Ar-2 locus.Finally,the embryos treated by PE3 were transferred to female mice,and 30(Hoxd13-1)and19(Hoxd13-2)mice were obtained,with the editing efficiencies of 26.7%(8/30)and 10.5%(2/19),respectively,and no off-target editing was detected by targeted deep sequencing and whole genome sequencing.2.The development and validation of ePEIn view of the low efficiency of PE in cells and embryos,the base complementary between the PBS and spacer of peg RNA,which would impair the targeted cleavage of DNA by Cas9/peg RNA complex,was proved by RNA structure prediction tools and experiments,and the fusion of Csy4 protein recognition site to the 3′ end of peg RNA(termed extended peg RNA)could inhibit this complementary.On this basis,the ePE was constructed by stepby-step optimizations including the fusion of extended peg RNA and nicking sg RNA,the fusion of Csy4 and PE2 protein and the point mutation on scaffold.For point mutations,the efficiency of ePE was tested in in HEK293 T,He La and mouse N2 a cells and compared to thePE3,and the results showed average increases of 1.9,3.8 and 4.9 times,respectively.For the insertions and deletions,ePE increased the prime editing efficiency by 1.2 and 0.6 times in HEK293 T cells,respectively.In addition,the length of RTT did affect the efficiency of ePE and ePE outperformed PE3;and ePE would not increase the off-target effects while improving the efficiency,but result lower outcome purity than PE3.3.The g.A746 G mutation in BMPR1 B gene mediated by ePE in sheep cellsTo induce the g.A746 G mutation into BMPR1 B gene of sheep,plasmids encoding PE3 and ePE were introduced into sheep fetal fibroblasts by electroporation.The results showed that the editing efficiency of PE3 was 8.9% with the indels frequency of 1.8%,while the editing efficiency of ePE was 57.3%,which was 6.4 times as high as that of PE3,with the indels frequency of 5.6%.The outcome purity of ePE was significantly higher than that of PE3,and the scaffold integration caused by both editors was at a very low level(0.02% and 0.19%,respectively).Furthermore,neither PE3 nor ePE induced non-target editing in sheep fetal fibroblasts,and off target editing caused by them was not detected,verifying the security of PEs.In summary,this study demonstrated the effectiveness of PE in mammals,laying foundation for its application in large animals;and developed a new prime editor,ePE,providing an efficient tool for animal breeding;and the ePE was applied to the editing of sheep BMPR1 B gene g.A746 G,providing technical support for the germplasm creation and biological breeding of livestock.