Generation Of CRISPR-mediated TBXT And FecB Gene Editing In Tan Sheep

Gene editing technology,represented by CRISPR/Cas9,is a genetic engineering technique that modifies the bases of target sequences or loci of genomic DNA.2019,researchers have developed a newest gene editing tool based on CRISPR/Cas9 system-Prime editing(PE)system.The system fuses the n Cas9 protein with a reverse transcription element and improves on the sg RNA to obtain a peg RNA(prime editing guide RNA),which incorporates a PBS(primer binding)sequence with a "primer" binding.The peg RNA combines a PBS(primer binding site)sequence with a RT(Reverse transcriptase)sequence that contains information about the editing site and can be written into the genome by reverse transcription.Compared with the base editor,this system can achieve any type of mutation between bases and efficient insertion and deletion of small fragment sequences.Therefore,the bootstrap editing system is of great importance in the construction of large animal disease models and improvement of animal economic traits.The Tan sheep is an important local sheep breed in China,and the dipterocarp it produces is the treasure of the Ningxia region,while it is loved by people nationwide because of its fine meat quality and superior flavor.However,the shortcomings of the Tan sheep,such as low fertility and fatty fat tails,have seriously restricted the development of the Tan sheep industry.Therefore,this study aimed to edit genes related to reproduction and tail vertebrae traits in Tan sheep by CRISPR/Cas9 system as well as a bootstrap editing system to lay a solid foundation for creating a high-quality Tan sheep breeding popμLation.The experimental procedure and main resμLts are as follows.(1)Preparation of TBXT gene editing Tan sheep by CRISPR/Cas9The sg RNA was designed and transcribed in vitro for the target site of TBXT gene(c.G334T),and a ss ODN was synthesized to achieve HDR editing.13 breeding females were selected and ovμLated in sheep farms,and 151 embryos were obtained,141 of which were injectable embryos with good morphology.The resμLts of Sanger sequencing showed that the TBXT gene was detected in 3 lambs.Sanger sequencing showed that the TBXT gene was edited in 3 lambs(25%)but did not produce base substitutions at the target site.Therefore,this study was followed by an attempt to construct TBXT point mutant Tan sheeps using a bootstrap editing system.(2)Preparation of TBXT gene edited Tan sheep using the prime editing systemSuitable peg RNAs and nick sg RNAs were designed for the target site of TBXT(c.G334T)and transcribed in vitro.Thirteen suitable breeding Tan sheep ewes were selected for supernumerary ovμLation and fertilized eggs were obtained.Using microinjection technique,100 ng/μL of PE2 m RNA,40 ng/μL of TBXT-peg RNA,5 ng/μL of TBXT-nick1 and 5 ng/μL of TBXT-nick2 microfractions were injected into 140 morphologically intact fertilized eggs,and 138 of these well-developed embryos were transferred into 24 recipient ewes.Eight lambs were obtained after 150 days.Deep target sequencing showed that three lambs had undergone targeted base editing(37.5%)and no off-target variants were detected genome-wide.(3)Preparation of FecB gene-edited Tan sheep using a prime editing systemPeg RNAs and nick sg RNAs were designed for the target site(g.A746G)of FecB,and the editing efficiency of peg RNAs and nick sg RNAs was initially verified at the cellμLar level.Twelve Tan sheep suitable for breeding ewes were selected for supernumerary ovμLation treatment and fertilized eggs were obtained,and 100 ng/μL of PE2 m RNA,40ng/μL of FecB-peg RNA and 5 ng/μL of FecB-nick microfraction were microinjected into128 morphologically and structurally intact fertilized eggs to obtain 124 intact embryos,which were transplanted into 21 recipient ewes and normal Seven lambs were obtained after normal gestation.Deep target sequencing-based assays showed that base editing occurred in five of the lambs(71.4%),while no off-target variants were detected at genome-wide scale.In summary,in this study,we firstly constructed TBXT gene editing Tan sheep using the CRISPR/Cas9 system and the prime editing system,both of which coμLd achieve TBXT gene editing.However,the CRISPR/Cas9 gene editing system only achieved gene knockout in the TBXT Tan sheep,while the prime editing system coμLd accurately achieve point mutation at the target site without causing any side editing(bystander)or off-target mutation,but the mutation frequency at the target site was low.In order to further confirm the effectiveness of the prime editing system,the FecB gene editing Tan sheep were prepared using the system,and the resμLts were similar to the previous ones,with high accuracy of the created FecB gene editing Tan sheep but low mutation frequency.The present experiment demonstrated that the bootstrap editing system can be accurately edited on large mammals,but the system still needs further optimization.