Generation Of HACE2 Gene-edited Pigs By CRISPR/Cas9-mediated Knock-in Strategy And Identification

A massive outbreak of 2019 coronavirus disease(Novel coronavirus pneumonia,COVID-19)from the end of 2019 poses a huge threat to people’s lives,health and economic livelihood.Although the novel coronavirus pneumonia has been classified as a class B infectious disease in China,because the virus mutates quickly and the effective time of the vaccine is only 6 months,and the infected cases appeared in May,and the strains have changed from the ones at the end of last year.However,the possibility of subsequent large-scale epidemics is not large,and there will be no impact on the medical order and social operation in the short term.There is a lack of specific drugs against Severe Acute Respiratory Syndrome Coronavirus 2(SARSCo V-2),and preventive measures are mainly based on vaccination.Therefore,At present,the effective response to COVID-19 still faces a serious challenge.Animal models of SARS-Co V-2 infection have an important role in the development of vaccines and potent drugs for neostriatal pneumonia.Animal models currently used for vaccine and drug development against neointimal pneumonia include macaques,cats,ferrets,hamsters,and transgenic mice,but to date,these animal models only partially mimic the clinical features of human neointimal pneumonia.Therefore,there is an urgent need to develop more effective animal models to improve the current deficiencies in research on the development of potential vaccines or drugs for the treatment of neostriatal pneumonia.Current studies have shown that angiotensin-converting enzyme 2(ACE2)plays an important role in mediating viral invasion of the host by interacting with the stinger glycoprotein of SARS-Co V-2.The spiky glycoprotein S protein of novel coronavirus SARS-Co V-2 binds to host cells through its receptor binding domain RBD,mediating the adhesion of virions to host cells to initiate infection.The susceptibility of different animal ACE2 to SARS-Co V-2 was different.CRISPR gene editing is a new technology that has been rapidly developed in recent years,and it has been successfully constructed in mice,zebrafish,rabbits,pigs and sheep,among others,have been successfully constructed in a variety of gene editing animal models.Currently,h ACE2 gene edited mice have been established,but with the defects in the species differences,this model cannot fully mimic the respiratory related symptoms produced by human infection with the virus,and there are limitations in vaccine development and drug applications.Because pigs have more similar anatomical,physiological,and immunological characteristics to humans,they are considered to be good animal models for human diseases.In order to prepare a transgenic pig model susceptible to SARS-Co V-2,we first constructed a targeting vector for human ACE2 gene replacement in vitro.The h ACE2 gene was site-specifically integrated into the Rosa26 site of the pig genome by CRISPR / Cas9-mediated site-specific integration technology,and a gene-edited PK-15 cell line expressing both porcine ACE2 and human ACE2 was successfully screened.The expression of h ACE2 m RNA and protein in the cells was detected.The results showed that h ACE2 could be stably and efficiently expressed in the cell line.Then,a pseudovirus packaging and infection system with HIV-1 genome as the core was established,and the susceptibility of the gene editing cell line to SARS-Co V-2 was evaluated by the SARS-Co V-2 pseudovirus infection system.The results showed that the knock-in of the receptor gene could increase the susceptibility of cells to pseudovirus.Finally,with the help of site-specific integration technology,somatic cell nuclear transfer technology and embryo transfer technology,h ACE2 knock-in gene editing cloned pigs were prepared,and piglet tail tip fibroblasts and various tissues and organs were isolated for gene expression identification.Finally,we successfully obtained two gene-edited cloned piglets,and h ACE2 was stably expressed.In summary,we successfully obtained h ACE2 overexpressed genetically modified pigs using the CRISPR/Cas9 system.The high expression of h ACE2 was verified at the cellular and individual levels,and the susceptibility of h ACE2 overexpressing PK-15 cells was verified at the cellular level using pseudovirus infection assays.It further enriched the new crown animal model and laid a foundation for research in this area.