Research On The Effectiveness Of Base Editors In Cattle Embryos

In recent decades,the yield of dairy cows has continued to increase,while the fertility has been declining.Highly selective breeding not only improves the yield of dairy cows,but also increases the degree of group inbreeding.With the increase in the degree of inbreeding in dairy cow populations,the probability of homozygous recessive harmful genes in individual dairy cows increases,and the risk of genetic defect diseases also increases.The large use of a limited number of bulls in cattle breeding has led to the rapid spread of recessive genetic diseases.Among the common genetic defects in bull pedigrees,there are pathogenic mutations caused by multiple base mutations.These genetic defects lead to the death of cattle embryos,miscarriage of cows or calf deformities,which brings huge economic losses every year.Base editors are derived from CRISPR/Cas9 gene editing system.They can be used to achieve precise base editing without DNA double-strand breaks,homologous templates and homology directed repair pathways.It is a common method to develop base editors that forming a fusion protein by linking Cas9 nickase or dead Cas9 protein,DNA single strand deaminases and proteins that can control repair pathways.At present,the base editors commonly used are divided into two classes:cytosine base editors and adenine base editors.Cytosine base editors can be used to achieve transition of C:G to T:A by cytosine deamination and DNA repair while adenine base editors can be used to achieve transition of A:T to G:C by adenine deamination and DNA repair.In this study,we obtained bovine fertilized eggs through in vitro culture and in vitro fertilization,and then used the cytosine base editor BE3 and adenine base editor ABE7.10 to realize high-efficiency gene editing in bovine embryos through microinjection for the first time.We confirmed the feasibility of using a base editor to directly perform gene editing in bovine embryos.At the same time,we used the targeted next-generation sequencing method to explore the off-target situation when using the BE3 system and ABE7.10 system for base editing in bovine embryos.The results of the study showed that there were off-target phenomenons near the on-target sites,but no obvious off-target phenomenon was found in the six predicted potential off-target sites.After that,we used the BE3 system and the ABE7.10 system to perform multi-gene editing in bovine embryos through microinjection,and confirmed the feasibility of using the base editor to perform multi-gene editing in cattle embryos at the same time.Finally,we used the BE3 system to knock out the CDX2 gene in cattle embryos through microinjection,and the results of immunofluorescence experiments showed that the knockout was successful.Through this research,we have achieved efficient base editing,multiple gene editing,and gene knockout in cattle embryos,confirming the huge potential of base editors for precise gene editing in cattle embryos,which is of great significance to carry out genetic defect repair and trait improvement in breeding cattle and provides a new idea for reducing embryonic death,miscarriage and calf deformity caused by genetic defects,and for reducing the resulting economic loss.