Improve Meat Production And Virus Resistance By Simultaneously Editing Multiple Genes In Livestock Using Cas12i^Max

Precision breeding is a technology that uses molecular biology techniques to achieve direct selection and efficient combination of genes,and to significantly improve breeding efficiency and breeding cycle.China is a large livestock country,and various meat proteins play an important role in the national economy,while China’s meat production is insufficient and partly relies on imports.How to apply molecular precision breeding to meet the rising demand of domestic meat consumption has been the focus of research in the field of livestock breeding in China.With the development of gene editing tools,the research on livestock improvement using gene editing technology has entered a high speed development period.As scientists continue to study gene editing tools,more and more gene editing tools have been published.Cas12i^Max,a gene editing tool modified from wild-type Cas12i by protein synergy technology（MIDAS）,has shown powerful DNA editing ability on human 293T cells with a wide range of PAMs sequences,enriching the gene editing toolbox.However,validation of the efficiency and function associated with Cas12i^Max has not been reported.In this study,we aimed at the improvement of traits in livestock animals,on the one hand,we verified the editing efficiency of Cas12i^Max in multiple species and multiple gene loci,on the other hand,we prepared a multi-gene editing multi-trait improvement local pig and cattle model,and initially verified the growth rate,muscle yield and the effect of multi-gene editing pig model on porcine blue ear virus PRRSV,porcine epidemic gastroenteritis virus TGE.The growth rate,muscle yield and resistance to porcine blue ear virus PRRSV and porcine epidemic gastroenteritis virus TGEV were initially verified.The main research contents and results are as follows.1.Cas12i^Max maintains efficient editing ability at the cellular level at multiple loci in multiple species of domestic animalsThe multi-locus targeting plasmid construction and transfection experiments of Cas12i^Max in porcine,bovine and sheep fibroblasts revealed that Cas12i^Max has comparable or higher indel efficiency than Cas9;in the tests of different PAM sequences,it was verified that it has a wide range of PAM,with the highest efficiency of 5`-TTN reaching about 72%;further multi-species statistics of indel information at multiple loci revealed that Cas12i Max DNA editing tends to produce larger deletions（>20bp）,a property that may play a greater role in gene knockdown.2.A multi-trait improved pig model was prepared by combining somatic cell cloning of pigsMonoclonal cell screening of porcine fetal fibroblasts was performed using Cas12i^Max,and the cr RNAs we used were ANPEP-cr RNA with an indel efficiency of:（81.82%）,CD163-cr RNA with an indel efficiency of:（57.14%）,and MSTN-cr RNA with an efficiency of:（46.67%）.Then 111 monoclonal cell lines were screened and identified,and finally one cell line was confirmed as a donor for somatic cell cloning,which had no detectable off-target or chromosomal variants due to multiple gene editing.1089cloned embryos were obtained by somatic cell cloning,and these embryos were transferred to four recipient sows,two of which became pregnant,both of which expired and produced seven piglets.Further verification of genotype and protein expression confirmed that the ANPEP,CD163,MSTN and IGF2genes were edited and protein expression was as expected in our ACMG pig model.In summary,we successfully prepared a multi-gene editing pig model using Cas12i^Max multiplasmid transfection combined with somatic cell cloning of pigs.Continuous examination of the birth model pig weight data revealed that the ACMG pig model was significantly heavier than the control compared to the wild-type pigs of the same age,and its average daily growth and meat ratio were also significantly higher than the wild-type pigs of the control group.HE staining of muscle and subcutaneous fat of the model pigs revealed that the cross-sectional area of muscle fibers was significantly higher in the model pigs than in the control wild-type pigs.Once again,it was demonstrated that editing of IGF2 and MSTN affected muscle yield and growth rate in the model pigs.The results of simulated virus infection experiments on WT and multi-trait modified ACMG pig models showed that we consistently detected positive PRRSV antibodies from 14 to 28 days after weak vaccination and also detected PRRSV pathogens in serum at 21 days after vaccination;we also detected positive TGEV antigens in serum from 4 to 10 days after vaccination and at 7 days.We also detected TGEV antigen positivity in serum at 4-10 days after vaccination and TGEV pathogen positivity at 7 days after vaccination,while we did not detect either virus antibody positivity or pathogen positivity in the ACMG model pigs that were vaccinated at the same time.This indicates that the ACMG model is resistant and less susceptible to the virus.The results of blood routine and blood biochemical indexes of ACMG pigs and wild-type pigs of the same age at 8 months of age showed that all 27 blood routine indexes were not different from wild-type.And 16 blood biochemical indicators also did not differ from the wild type.Further multi-histopathological analysis of the ACMG model and wild-type control pigs showed no abnormalities in individual body tissues and organs.3.Cell line screening and cloning of multi-gene edited cattleMonoclonal cell screening of fetal fibroblasts from Mongolian cattle was performed by multiplasmid transfection with Cas12i^Max,and the 89 pig strain cell lines screened were further characterized,and finally the multi-gene edited cell lines with MSTN,PRNP and CD18 single amino acid mutations were confirmed.After genotype confirmation and off-target assay and other quality checks on the cell lines,the next step of cloning was performed.The triple-edited bovine clone embryos developed normally and showed a 60%pregnancy rate by ultrasound 40 days after embryo transfer,which also tentatively demonstrated the potential of the triple-edited bovine embryos to develop into complete individuals.In summary,in this study,we prepared and phenotypically validated 4-gene edited pigs with high growth rate,high muscle yield and resistance to porcine blue ear virus（PRRSV）and epidemic diarrhea virus（TGEV）using Cas12i^Max;finally,we further prepared 4-gene edited pigs with high muscle yield and resistance to acute pneumonia caused by classical spongiform encephalopathy（BSE）and Mannheimia haemolytica infection.3 gene-edited Mongolian cattle.While the phenotypes of progeny bred using the multi-gene edited pig model and the phenotypes of multi-gene edited cattle need to be further investigated.Our study provides a new tool for gene editing breeding and a new direction for rapid multi-trait improvement of livestock.