Study On Mutations In F1 Gene-Edited Cashmere Goats Based On Trio-Based Sequencing

At present,CRISPR/Cas9 technology is the most widely used gene editing technology,and its safety evaluation is great significance for the future production and clinical application of this technology.In this study,the F1 population of Shaanbei Cashmere goats obtained by using the MSTN and FGF5 gene edited generated previously.The whole genome of gene-edited cashmere goats and their offspring(F1 generation)were sequenced,and de novo mutations were strictly filtered,and the true mutations caused by CRISPR/Cas9 system were judged by combining the prediction of off-target sites and the distribution of new mutations.These variations include: single nucleotide variants(SNVs),insertion-deletion(Indels)and structural variants(SVs).Subsequently,transcriptome sequencing was used to detect gene expression differences using the skin tissue of FGF5 edited goats and the muscle tissue of MSTN edited individuals as well as the WT.Finally,Immunofluorescence technique and Western blot were used to detect the changes of p53 protein in MSTN edited animals,in order to evaluate the potential risk of CRISPR/Cas9 system in the editing process.The main findings are as follows:1.Production of F1 goats.The F1 progeny of MSTN and FGF5 gene edited cashmere goats were generated by natural breeding.The genotype of the gene-edited cashmere goats was detected by Sanger sequencing.Except the #P59 individuals of F1 generation did not generated Indels caused by gene edited,indicating all others(#P6,#P97,#P8 and #P9)generated Indels by edited;2.Trio-based sequencing and variants detection.11 goats from four trios were sequenced,and detected the genetic relationship,so as to ensure that the trio information was consistent with the records of the genetic relationship during the breeding process.Specific SNVs of F1 generation were obtained through strict filtering procedures.In order to test the accuracy of filtration,Sanger sequence was used to verified these SNVs,with an accuracy of 70%.Meanwhile,in order to further determine whether these variants caused by gene edited,the trio-based sequencing was used to detect the distribution of these SNVs specific in F1 generation on genomes.A total of 19 indels(10 inserts and 9 deletions)were detected.Average 3.8 in F1;Four CNVs(2 duplications and 2 deletions)were detected,with an average of 1.5 CNVs per F1 individual.These results are the same as the number of variants per generation in normal trios.3.Off-target mutations in gene-edited goats.Two tools Cas-OT and Cas-OFFinder were used to predict the off-target sites corresponding to the four sgRNAs in MSTN and FGF5.After the overlap was taken,340,483,470 and 442 predicted off-target sites were obtained in the four sgRNAs,and none of the de novo SNV in the edited individuals appeared in the predicted off-target area.The results showed that the gene-edited did not cause off-target mutations associated with SNV.4.Transcriptomic analysis in F1.To better understand the transcriptional consequences of gene disruption in the genome of F1 progenies,we conducted transcriptome sequencing(RNA-seq)analysis in the edited progenies and WT animals using muscle or skin.Compared muscle tissues from MSTN edited individuals with WT animals,a total of 43 differentially expressed genes(DEGs)were detected,of which 23 were up-regulated and 20 were down-regulated,DEGs are known to be associated with muscle developmental.Compared skin tissues from FGF5 edited individuals with WT animals,a total of 140 differentially expressed genes were detected,of which 74 were up-regulated and 66 were down-regulated,DEGs are related to skin/hair follicle development.5.The p53 expression in edited goats.p53 may be related to tumor formation.Therefore,Immunofluorescence technique and Western blot were used in this study to detect changes in p53 genes expression in muscle tissues of MSTN-edited goats,and the results showed that there was no significant difference between the edited and unedited group.mRNA analysis showed that there was no significant change in the mRNA expression of genes related to p53 and programmed cell death between the edited and unedited group.This indicated that CRISPR/Cas9 technology did not introduce potential risks in the offspring of edited goats.In conclusion,no DSB induced SNVs were detected in the genome of gene-edited cashmere goats,meanwhile,the mutation rate of F1 is almost the same as that of normal trio.The results of mRNA show that DEGs in MSTN edited individuals muscle tissues were mainly related to the development of muscle fibers,while DEGs in FGF5 edited individuals skin tissues were mainly related to the development of skin,hair follicles and the immune system.Protein results showed that CRISPR/Cas9 did not induce significantly changed expression of p53 protein.It has preliminarily proved the reliability of gene editing technology and laid a foundation for the application of CRISPR/Cas9 technology in animal breeding and biomedicine.