Highly Efficient Generation Of Sheep With A Defined FecB Mutation Via Adenine Base Editing

In recent years,the excellent animal and plant germplasm materials and varieties created based on various gene editing tools,as well as the correction and treatment of some genetic diseases are full of expectations.The gene editing technology represented by the CRISPR system is constantly innovating and developing.At present,based on the third-generation gene editing technology(CRISPR),a single-base gene editing system is further derived.Generally,base editors can be divided into two types according to different types of deaminase fusion,namely cytosine base editors(CBEs)and Adenine base editors(ABEs).Without introducing double-strand break(DSB),the base editor can induce single-base substitution at a specific site by catalyzing the base deamination reaction.Compared with CRISPR-Cas9,the base editor has higher editing efficiency and less off-target effects.At present,several research groups at home and abroad have applied it to the gene editing of cells,animals and plants.Because genetic mutations related to many important economic traits of livestock are single-base mutations,single-base editors can be used as molecular breeding methods to introduce mutations at the desired target site.This laboratory used BE3 to successfully introduce point mutations into the SOCS2 gene of Tan sheep,and the editing efficiency was as high as 75%,indicating that it is feasible to use the base editor for gene editing in large mammals.This provides an example and technical basis for the application of ABEs in molecular breeding of livestock.The FecB gene is one of the major genes controlling the high fertility of sheep.This gene has the functions of inducing the differentiation of granulosa cells of ewe follicles,promoting the development of follicles,and increasing the number of ovarian ovulations.Studies have shown that point mutations of this gene(g.A746 G,p.Q249R)cause mutations in glutamine in highly conserved regions of bone morphogenetic protein receptors(Bone Morphogenetic Protein,BMP)to arginine.In the previous experiment,our laboratory used the CRISPR-Cas9 system to inject m RNA and single-stranded oligonucleotide(ss ODN)donor sequences into goat and sheep fertilized eggs,and successfully obtained the results of introducing the expected point mutations,but Editing efficiency is not high,24% and 23.8% respectively.The purpose of this experiment was to use in vitro transcription and microinjection techniques to introduce ABEs system into sheep 1-cell embryos,and to precisely modify the Tan sheep genome,and to use embryo transfer technology to obtain Tan sheep individuals carrying specific point mutations of the FecB gene.The test process and the main results obtained are as follows:(1)Verification and screening of multi-version ABEs editing effects at the cellular levelIn this experiment,a sg RNA was designed at the target site of the FecB gene.Using liposome transfection technology,multiple versions of ABEs were applied to sheep cell-level gene editing.Sanger sequencing and TA cloning were used to detect the editing efficiency to determine the type of gene editing.Cas-OFFinder software predicts and detects off-target sites of ABEs in the whole genome range,comprehensively evaluates them in various aspects,and then selects the optimal ABEs version.The experimental results show that the editing efficiency of ABE7.10,ABEmax and x Cas9-ABE on sheep FecB gene is 18.75%,53.85% and 38.46%,respectively.Compared with the other two base editors,ABEmax has the highest editing efficiency and no off-target phenomenon.Therefore,ABEmax can be used for sheep gene editing.(2)Verification and evaluation of ABEmax editing effect embryo levelUsing in vitro transcription and microinjection techniques,ABEmax m RNA and FecB-sg RNA were introduced into Tan sheep 1 cell stage embryos,and gene editing positive embryos were obtained by in vitro cultivation.Sanger sequencing and T-A clone were used to detect the editing efficiency to determine the type of gene editing;Cas-OFFinder software was used to predict the off-target site of ABEmax in the whole genome range and detect it.The test results showed that a total of 48 embryos were obtained in vitro,of which 10 embryos carrying the expected mutations were produced,and another embryo was produced for side editing.T-A clone test showed that ABEmax had better editing ability on Tan sheep embryos(efficiency 22.91%).Cas-OFFinder predicted a total of 5 off-target sites(OT1-OT5).The PCR products of 11 embryos predicted off-target sites were sequenced,and the results showed that no off-target phenomenon occurred.The experiment proves that ABEmax has a good effect of introducing point mutations on sheep embryos,and does not cause off-target effects in the whole genome.(3)Creation and evaluation of FecB gene mutation Tan sheepUsing the estrus and superovulation technique during the same period,96 1 cell embryos were obtained from 10 donor ewes,and the well-developed embryos were transplanted into 18 recipient ewes by microinjecting ABEmax m RNA and sg RNA.As a result,6 ewes became pregnant and 8 gene-edited lambs were finally obtained.After TA cloning,these 8 gene editing lambs successfully introduced point mutations,of which 6 lambs produced the expected base substitution(75%)at the p.Q249 R site,and 2 lambs only happened nearby Mutation(25%)without target site mutation.Deep sequencing shows that the maximum editing efficiency of ABEmax at the target site has reached 48.5%.The off-target detection found that no base mutation occurred at the five predicted sites where off-target might occur.This indicates that ABEmax has higher gene targeting accuracy at the individual sheep level,and no off-target phenomenon has occurred.In summary,this study used ABEs to successfully generate the expected FecB mutation in Tan sheep individuals.The editing efficiency of Sanger sequencing and T-A clone detection was used to determine the type of gene editing;Cas-OFFinder software was used to predict the possible off-target sites of ABE in the whole genome,and corresponding detection and comprehensive evaluation were carried out.It is the first time to prove that the ABE system is feasible and safe for molecular breeding of sheep gene editing.