CRISPR/Cas9-based Genome Editing In Porcine Fetal Fibroblasts And Generation Of Genetically Modified Pigs At MSTN Locus

Genetically-modified pigs play an important role in agricultural and biomedical fields.Porcine growth traits,disease-resistant performance and meat quality can be improved faster through genetic modifications.Furthermore,genetically-modified pigs are increasingly used as disease model and xenotransplantation donor,making a great contribution to human health.CRISPR/Cas9 is a newly-developed genome editing tool in which Cas9 nuclease generates DNA double-strand breaks at the specific site under the guidance of gRNA.Diverse mutation patterns could be induced in response to different DNA repair pathways.Due to the high efficiency and simple assembly,CRISPR/Cas9 has emerged as the most popular genome editing tool and has been successfully applied in many species such as Caenorhabditis elegans,Drosophila,Mice and Goat.MSTN,a member of the transforming growth factor-? superfamily,mainly functions as a negative regulator of muscle growth and development.MSTN-deficient mice showed significantly increased muscle mass resulting from myofiber hyperplasia and hypertrophy.In addition,several cattle breeds carrying natural MSTN mutations exhibited double-muscled phenotype characterized by enhanced muscle mass.All these observations have made MSTN a strong candidate gene for improving porcine growth performance.Genetic modification at porcine MSTN gene hence is promising for cultivating new breeds with a high lean meat rate.The present project focused on genome editing in porcine fetal fibroblasts.An efficient and flexible porcine genome editing platform was built based on the CRISPR/Cas9.Firstly,the transfection efficiency was significantly improved to 90% by optimizing electroporation parameters and media,thereby promoting genome editing mediated by Cas9/gRNA.We found that diverse mutation patterns could be induced at porcine MSTN locus including single Cas9/gRNA-mediated indels,dual Cas9/gRNA-mediated deletions or inversions,and ssODN-mediated HDR.The overall mutation rate induced by dual gRNAs exceeded 30%,much higher than that of individual gRNA.Later,we achieved efficient single-base substitutions by optimizing the dosage and length of ssODN templates.With a HDR efficiency of more than 10%,two pathogenic point mutations for Alzheimer's disease and Parkinson's disease were introduced into porcine APP and LRRK2 loci,respectively.Interestingly,incomplete HDR was found when identifying PFF colonies.Only partial point mutations close to the Cas9 cutting site were incorporated.We further investigated the effect of mutation-to-cut distance on incorporation rate by deep sequencing.The results suggested that the point mutation near the Cas9 cutting site exhibited a higher HDR efficiency than the one which is distant from the DSBs.Finally,we obtained selectable marker-free PFF colonies carrying diverse mutation patterns in MSTN locus via limited dilution.Several kinds of cloned pigs were generated by SCNT,including Large White harboring the C313 Y mutation in MSTN locus,MSTN knockout Landrace and Erhualian pigs.Knockout Landrace showed partial double-muscled phenotype such as intermuscular grooves,enlarged tongues and myofiber hyperplasia.On the other hand,knockout Erhualian pigs exhibited more obvious muscular protrusion and wider back and hip compared with wild-type cloned piglets.Furthermore,we examined these gene-edited pigs for exogenous transgene integration and off-target mutation.Collectively,this project has achieved optimization and application of a CRISPR/Cas9-based platform for generating genetically-modified pigs,thus facilitating the development of more precise disease modeling and genetic breeding in pigs.