Background Chinese indigenous pigs are popular with consumers for their juiciness,flavour and meat quality,but they have lower meat production.Insulin-like growth factor 2(IGF2) is a maternally imprinted growth factor...Background Chinese indigenous pigs are popular with consumers for their juiciness,flavour and meat quality,but they have lower meat production.Insulin-like growth factor 2(IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation.A single nucleotide polymorphism(SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor,zinc finger BED-type containing 6(ZBED6),leading to up-regulation of IGF2 and causing major effects on muscle growth,heart size,and backfat thickness.This favorable mutation is common in Western commercial pig populations,but absent in most Chinese indigenous pig breeds.To improve meat production of Chinese indigenous pigs,we used cytosine base editor 3(CBE3)to introduce IGF2 intron3-C3071T mutation into porcine embryonic fibroblasts(PEFs) isolated from a male Liang Guang Small Spotted pig(LGSS),and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer(SCNT) to generate the founder line of IGF2^(T/T) pigs.Results We found the heterozygous progeny IGF2^(C/T) pigs exhibited enhanced expression of IGF2,increased lean meat by 18%-36%,enlarged loin muscle area by 3%-17%,improved intramuscular fat(IMF) content by 18%-39%,marbling score by 0.75-1,meat color score by 0.53-1.25,and reduced backfat thickness by 5%-16%.The enhanced accumulation of intramuscular fat in IGF2^(C/T) pigs was identified to be regulated by the PI3K-AKT/AMPK pathway,which activated SREBP1 to promote adipogenesis.Conclusions We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality,and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3KAKT/AMPK signaling pathways.Our study provides a further understanding of the biological functions of IGF2and an example for improving porcine economic traits through precise base editing.展开更多
Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base edito...Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base editors offer the ability to induce precise nucleotide substitutions(Komor et al.,2016).Cytidine base editors(CBEs)consist of a cytidine deaminase fused with a Cas9-nickase variant(Cas9n,with a D10A substitu-tion)and can achieve site-specific C-to-T substitution.Similarly,adenine base editors use an adenine deaminase forA-to-G substi-tution.These systems have been used in various organisms(Mishra et al.,2019).However,the Cas9 complex requires target sites containing NGG protospacer adjacent motifs(PAMs),thus restricting selection of potential targets.A number of CBEs have been developed using Cas9 variants(mostly Cas9n),cytidine deaminases(such as rAPOBEC1 and PmCDA1),and uracil glycosylase inhibitor(UGI)domains.These CBEs of the first generation(BE1,rAPOBEC1-dCas9),second generation(BE2,rAPOBEC1-dCas9-UGI),and third generation(BE3,rAPOBEC1-Cas9n-UGI)have moderate editing efficiencies in mammalians(Komor etal.,2016).展开更多
基金supported by the National Natural Science Foundation of China (3207269732030102)+2 种基金CARS-PIG-35R&D Programmes of Guangdong Province (2018B020203003)Laboratory of Lingnan Modern Agriculture Project (NZ2021006)。
文摘Background Chinese indigenous pigs are popular with consumers for their juiciness,flavour and meat quality,but they have lower meat production.Insulin-like growth factor 2(IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation.A single nucleotide polymorphism(SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor,zinc finger BED-type containing 6(ZBED6),leading to up-regulation of IGF2 and causing major effects on muscle growth,heart size,and backfat thickness.This favorable mutation is common in Western commercial pig populations,but absent in most Chinese indigenous pig breeds.To improve meat production of Chinese indigenous pigs,we used cytosine base editor 3(CBE3)to introduce IGF2 intron3-C3071T mutation into porcine embryonic fibroblasts(PEFs) isolated from a male Liang Guang Small Spotted pig(LGSS),and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer(SCNT) to generate the founder line of IGF2^(T/T) pigs.Results We found the heterozygous progeny IGF2^(C/T) pigs exhibited enhanced expression of IGF2,increased lean meat by 18%-36%,enlarged loin muscle area by 3%-17%,improved intramuscular fat(IMF) content by 18%-39%,marbling score by 0.75-1,meat color score by 0.53-1.25,and reduced backfat thickness by 5%-16%.The enhanced accumulation of intramuscular fat in IGF2^(C/T) pigs was identified to be regulated by the PI3K-AKT/AMPK pathway,which activated SREBP1 to promote adipogenesis.Conclusions We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality,and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3KAKT/AMPK signaling pathways.Our study provides a further understanding of the biological functions of IGF2and an example for improving porcine economic traits through precise base editing.
基金grants from the Major Program of Guangdong Basic and Applied Research(2019B030302006)the National Natural Science Foundation of China(31921004+1 种基金31971915)the Guangdong special support program of Young Top-Notch Talent in Science and Technology Innovation(2019TQ05N147).
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base editors offer the ability to induce precise nucleotide substitutions(Komor et al.,2016).Cytidine base editors(CBEs)consist of a cytidine deaminase fused with a Cas9-nickase variant(Cas9n,with a D10A substitu-tion)and can achieve site-specific C-to-T substitution.Similarly,adenine base editors use an adenine deaminase forA-to-G substi-tution.These systems have been used in various organisms(Mishra et al.,2019).However,the Cas9 complex requires target sites containing NGG protospacer adjacent motifs(PAMs),thus restricting selection of potential targets.A number of CBEs have been developed using Cas9 variants(mostly Cas9n),cytidine deaminases(such as rAPOBEC1 and PmCDA1),and uracil glycosylase inhibitor(UGI)domains.These CBEs of the first generation(BE1,rAPOBEC1-dCas9),second generation(BE2,rAPOBEC1-dCas9-UGI),and third generation(BE3,rAPOBEC1-Cas9n-UGI)have moderate editing efficiencies in mammalians(Komor etal.,2016).
