Genome Editing At MSTN And CLPG1 Locus In Rabbit And Goat Using Cas9

China is abundant in indigenous goat and rabbit species,however specialized breed for meat are in short,which makes the improvement of the meat productivity of indigenous breeds in urgent.MSTN and CLPG1 are the most well-known genes that promote muscle development up to now,and their mutations results in the "Double Muscled(DM)phenotype" and "Callipyge(CLPG)phenotype" respectively,both phenotypes improve the muscle weight and dressing percentage.Until now,no polymorphisms of MSTN and CLPG1 in goat and rabbit was found to cause DM phenotype and CLPG phenotype.Mutations of MSTN and CLPG1 can be generated by genome editing technology such as CRISPR/Cas9,and which is a promising way to improve the meat productivity of goat and rabbit.Cas9,guided by single strand RNA,is an easy-handle,highly specific,efficient,and multiplexable approach for engineering eukaryotic genomes.In this study,we utilized Cas9 to generate MSTN KO and CLPG1 KO rabbits and goats,and further evaluated the phenotywpe changes.This study contained the following four parts;1.Generation of MSTNKO rabbits using Cas9 systemTwo sgRNAs,sgRl and sgR2 targeted rabbit MSTN exonl and exon3 respectively were chosen,and their efficiencies in one-cell stage rabbit embryos after cytoplasm injection were up to 62.5%(5/8)and 66.7%(4/6)respectively.Next,315 injected embryos were transferred to 27 recipient rabbits,12 of them carried full term pregnancies and delivered 32 rabbits.During the 32 newborn rabbits,24(75%)were edited,59.4%(19/32)of them were mutated at each of the target site respectively,and 43.8%(14/32)mutated at both the target sites.As expected,the birth weight of the MSTN KO rabbits were higher than that of normal rabbits,and the weight ratio of tongue,biceps,quadriceps to whole body were significantly higher than that of the control rabbits.MSTN protein level in longissimus muscle and tongue muscle decreased 60%and 55%respectively and the myogenin expression increased about 50%in longissimus muscle.However,unexpected higher death ratio(50%,12/24)of the MSTN KO rabbits at birth and the first week(early stage)were found in the MSTN KO rabbits.Moreover 58.3%(14/24)of the MSTN KO rabbits showed enlarged tongue.Both the phenomenon were absent in the control and MSTN WT rabbits.These results indicated that MSTN KO causeed muscularity,however it also caused the health problems such as high early stage death rate and large tongue phenomenon.These side effects of MSTN KO should be carefully studied in the future,especially in F1 and F2 of MSTN KO rabbits before the MSTN KO strategy could be applied in the farm area.2.Generation of MSTN KO goats using Cas9 systemsgG targeted the exon 3 of goat MSTN was chosen.Goat embryos were injected to with different amouts of Cas9 mRNA and sgG,low concentation group(50 ng/μL and 10 ng/μL)and high concentation group(100 ng/μL and 50 ng/μL)respectively.Six and eight recipients were transferred with injected embryos from each group,and 3 and 4 goats were pregnant and delivered 4 and 6 lambs.25%of the goats(1/4)from low concentration and 66.7%(4/6)group from high concentration group were MSTN edited.Among the 5 edited lambs,one were born with enlarged tongue,however it disappeared at 2 months old.In contrast to high death rate in MSTN KO rabbits,no stillborn phenomenons were found in the edited lambs.3.Generation of CLPG1 KO rabbits using Cas9 systemFirst,by searching the 12 bp conserved motif of CLPG1 at the 3’-sequences of DLK1 and multi-alignment of 400 bp CLPG1 sequences from different species,rabbit CLPG1 was located at 48 kb downstream of DLK1.Then two sgRNAs(rCLPG_sgDu and rCLPG_sgDd)were chosen.In rabbit embryos,both sgDu and sgDu+sgDd injected embryos(5 and 12 embryos respectively)showed 100%genome editing efficiencies.For embryos transfer,5 recipients received 78 embryos after injecting with both sgRNAs,7 rabbits were delivered from two pregnancies.All the 7 rabbits were found to be edited at the CLPG1 locus,and most of the mutations were indels which means CLPG1 were KO.This is the first study to generate CLPG1 KO animals,and will facilitate the study of CLPG phenotype.4.Generation of CLPG1 KO goat using Cas9 systemTo detect the polymorphisms,493 bp of goat CLPG1 sequence was amplified by Direct-PCR kits using goat blood as templates.CLPG1 c.414 A>C and rare SNP c.448 T>A rather than sheep CLPG1 c.267 A>G were found,and c.414 A>C were in Hardy-Weinberg balance.Next,two sgRNAs(gCLPG_sgland sgCLPG_sg2)targeted goat CLPG1 locus were chosen and injected to goat embryos with Cas9 mRNA,and 47 embryos out of 56 injected embryos were transferred to 22 recipient goats,finally 15 lambs were delivered from 13 pregnancies.66.7%(9/15)lambs were edited with indels,which means goat CLPG1 were KO.This is the first study to generate CLPG1 KO goats,and will facilitate the research of epigenetics regulation in DLK1-DIO3.