Development of humanized ACE2 mouse and rat models for COVID‐19 research

Abstract

To date (17 November 2021), there has been more than 254 million confirmed cases of COVID‐19, and more than 5 million death globally (World Health Organization. https://covid19.who.int/). The virus causing COVID‐19 is called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). SARS‐CoV‐2 infects host cells by the binding of its spike protein to the cellular surface protein angiotensin‐converting enzyme 2 (ACE2). The predicted 29 amino acid residues of ACE2 that interact with SARS‐CoV‐2 spike protein receptor binding domain (RBD) vary between human ACE2 and mouse or rat ACE2. Therefore, wildtype mice and rats show lower SARS‐CoV‐2 infection rate and mild symptoms compared to what is seen in humans. Small animal models that recapitulate human COVID‐19 disease are urgently needed for better understanding the transmission and therapeutic measurement. Currently, scientists use either mouse‐adapted SAS‐CoV‐2 (SAS‐CoV‐2 MA) models or random transgenic mouse models that artificially express human ACE2 under the control of cytokeratin 18 promoter or a constitutive promoter. SAS‐CoV‐2 MA may not completely reflect all aspects of the original human‐tropic SAS‐CoV‐2 and the current transgenic human ACE2 mouse models typically have high mortality rate caused by neuroinvasion and encephalitis due to very high human ACE2 expression. To overcome these limitations, we have developed humanized ACE2 mouse and rat models using CRISPR‐Cas9. Specifically, we inserted a ~3kb human ACE2 cDNA cassette into the mouse and rat Ace2 gene loci to ensure that human ACE2 expression is under the control of rodent Ace2 promoter and regulatory elements, while simultaneously disabling the rodent Ace2 gene. To accomplish this, CRISPR gRNAs targeting close to the translation initiation site of Ace2 were screened in cultured mouse and rat cells. Then CRISPR/Cas9 complex and donor DNA were subsequently microinjected into one‐cell stage embryos which were subsequently implanted into pseudo pregnant females. Resulting pups were screened for correct knockin by junction PCR and insert PCR, and the PCR products were Sanger sequenced. Targeted Locus Amplification (TLA) further confirmed the integration sites and transgene sequence. RT‐qPCR and Western blot analysis data showed that, in our models, human ACE2 is expressed in tissues expressing endogenous Ace2 (such as lung, kidney, and GI tract), while rodent endogenous Ace2 is absent from these tissues. Further breeding data indicated that both hemizygous and homozygous humanized ACE2 animals appear to be normal and fertile. Most importantly, animals displayed symptoms after infection with SARS‐CoV‐2. In summary, these data suggest that our humanized ACE2 models can be valuable for COVID‐19 research.