Dual SgRNAs Facilitate CRISPR/Cas9 Mediated Genome Engineering And Animal Model Generation

Since 2012,the bacterial CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9)immune defense system,utilized as a RNA-guided endonuclease,has been shown to be a mammalian genome engineering tool,and by co-microinjection of Cas9 mRNA and sgRNAs into one-cell stage embryos,mouse genome targeting and modeling can be achieved easily in one driect step.Here,we used dual sgRNAs strategy for targeting one single gene with the CRISPR/Cas9-mediated genome targeting efficiency to 100%and bi-allelic modification efficiency up to 78%,bi-allelic frameshifted mutation of ApoE was verified by function analysis.And CRISPR/Cas9 can induce site-dependent off-target effects in the mouse cell line and zygotes.Target gene modifications can be passed to the next generation through germline transmission.So the dual sgRNAs strategy facilitates CRISPR/Cas9-mediated mouse genome engineering.Taking advantage of high efficiency of dual sgRNAs strategy and the multiplexable feature to target genome of the CRISPR/Cas9 system,we explored the applicability and effectivity of multiple genes targeting.By zygote co-microinjection of Cas9 mRNA and multiple pairs of sgRNAs targeting immune-associated genes B2m,Il2rg,Prfl,Prkdc,and Rag1,we successfully achieved one step generation of immunodeficient mouse models.The established founder mice harbored varieties of gene modifications and fragment deletion,especially double knockout of B2m and Ragl which are on the same chromosome,resulting different kinds of immunodeficient mouse models with different combinations of genetic modifications.The application of dual sgRNA strategy shows time-and labor-saving economic advantages over previous methods in generating multiple gene deficiency mouse models.Based on the characterization and application of dual sgRNA strategy for genome editing on model organism,we investigated the applicability and efficiency of this method in the large farm animal goat.We successfully got the gene modified goats which contains one or both allels disrupted,by co-microinjection of one-cell stage zygotes with Cas9 mRNA and sgRNA targeting the functional gene(MSTN).In the cultured fibroblasts,the MSTN targeting efficiency was as high as 60%,while in the 79 alive animals,the efficiency of modifying MSTN was 12.7%.The on-target modifications and off-target mutations of MSTN in fibroblasts,as well as in somatic tissues,especially the muscles and sex gonads of founder goats,were carefully analyzed,indicating that CRISPR/Cas9 mediated MSTN disputation can be achieved in large animals.The phenotypes of MSTN-disrupted goats by CRISPR/Cas9 remain to be evaluated extensively.Through body mass recording,H&E staining,TEM,quantitative PCR,Western blotting and immunofluorescence staining,we demonstrated that MSTN-modified goats got increased body weight and larger size of muscle fibers.By whole genome amplification of germ cell and PCR,we further demonstrated that knockout NSTN alleles existed in the germ cells,and were highly germline transmittable.These results demonstrated that using CRISPR/Cas9 system for establishment of genome modified livestock with desirable phenotype is applicable.Together with other investigations of CRISPR/Cas9 mediated gene modification in other livestock species such as pigs and dogs,our results demonstrate that the CRISPR/Cas9 system is a robust and efficient gene editing tool in farm animals,and therefore there will be a great application prospect for livestock breeding.In conclusion,results from cell line,mice and goats demonstrate the CRISPR/Cas9 based dual sgRNA strategy can facilitate genome engineering and animal generation.