Elimination of GGTA1,CMAH,β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

Background:Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic,while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs.Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection(HAR)that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure,in which process the MHCⅡmolecule plays critical roles.Methods:Thus,we generate a 4-gene(GGTA1,CMAH,β4GalNT2,and CIITA)knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.Results:We successfully obtained 4KO piglets with deficiency in all alleles of genes,and at cellular and tissue levels.Additionally,the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping.Piglets have survived for more than one year in the barrier,and also survived for more than 3 months in the conventional environment,suggesting that the piglets without MHCⅡcan be raised in the barrier and then gradually mated in the conventional environment.Conclusions:4KO piglets have lower immunogenicity,are safe in genomic level,and are easier to breed than the model with both MHCⅠandⅡdeletion.

Long-term effects of gravel―sand mulch on soil organic carbon and nitrogen in the Loess Plateau of northwestern China

Gravel-sand mulch has been used for centuries to conserve water in the Loess Plateau of north- western China. In this study, we assessed the influence of long-term （1996-2012） gravel-sand mulching of cultiv- ated soils on total organic carbon （TOC）, light fraction organic carbon （LFOC）, microbial biomass carbon （MBC）, total organic nitrogen （TON）, particulate organic carbon （POC）, mineral-associated organic carbon （MOC）, perma- nganate-oxidizable carbon （KMnO4-C）, and non-KMnO4-C at 0-60 cm depths. Mulching durations were 7, 11 and 16 years, with a non-mulched control. Compared to the control, there was no significant and consistently positive effect of the mulch on TOC, POC, MOC, KMnO4-C and non-KMnO4-C before 11 years of mulching, and these organic C fractions generally decreased significantly by 16 years. LFOC, TON and MBC to at a 0-20 cm depth in- creased with increasing mulching duration until 11 years, and then these fractions decreased significantly between 11 and 16 years, reaching values comparable to or lower than those in the control. KMnO4-C was most strongly correlated with the labile soil C fractions. Our findings suggest that although gravel-sand mulch may conserve soil moisture, it may also lead to long-term decreases in labile soil organic C fractions and total organic N in the study area. The addition of manure or composted manure would be a good choice to reverse the soil deterioration that occurs after 11 years by increasing the inputs of organic matter.

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i^(Max)

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated gene(Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i^(Max), a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i^(Max)in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163,and MSTN via Cas12i^(Max)in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i^(Max)for gene editing in livestock animals and demonstrated the potential application of Cas12i^(Max)in the field of animal trait improvement for agricultural production.

Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs

Blastocyst complementation by pluripotent stem cell(PSC)injection is believed to be the most promising method to generate xenogeneic organs.However,ethical issues prevent the study of human chimeras in the late embryonic stage of development.Primate embryonic stem cells(ESCs),which have similar pluripotency to human ESCs,are a good model for studying interspecies chimerism and organ generation.However,whether primate ESCs can be used in xenogenous grafts remains unclear.In this study,we evaluated the chimeric ability of cynomolgus monkey(Macaca fascicularis)ESCs(cmESCs)in pigs,which are excellent hosts because of their many similarities to humans.We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos,in which domesticated cmESCs(D-ESCs)injected into pig blastocysts differentiated into cells of all three germ layers.In addition,we obtained two neonatal interspecies chimeras,in which we observed tissue-specific D-ESC differentiation.Taken together,the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model,with a chimeric ratio of 0.001-0.0001 in different neonate tissues.We believe this work will facilitate future developments in xenogeneic organogenesis,bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.

Single-cell RNA Sequencing Reveals Thoracolumbar Vertebra Heterogeneity and Rib-genesis in Pigs

Development of thoracolumbar vertebra(TLV)and rib primordium(RP)is a common evolutionary feature across vertebrates,although whole-organism analysis of the expression dynamics of TLV-and RP-related genes has been lacking.Here,we investigated the single-cell transcriptome landscape of thoracic vertebra(TV),lumbar vertebra(LV),and RP cells from a pig embryo at 27 days post-fertilization(dpf)and identified six cell types with distinct gene expression signatures.In-depth dissection of the gene expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and RP development.Further analysis of cell type-specific and strand-specific expression uncovered the extremely high level of HOXA103′-UTR sequence specific to osteoblasts of LV cells,which may function as anti-HOXA10-antisense by counteracting the HOXA10-antisense effect to determine TLV transition.Thus,this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell type-specific resolution,which serves as a comprehensive view on the transcriptional profile of animal embryo development.