Nonsense mutations that lead to the insertion of a premature termination codon (PTC) in the cystic fibrosis transmembrane conductance regulator (CFTR) transcript affect 11% of patients with cystic fibrosis (CF) worldwide and are associated with severe disease phenotype. While CF rat models have contributed significantly to our understanding of CF disease pathogenesis, there are currently no rat models available for studying CF nonsense mutations. Here we created and characterized the first homozygous CF rat model that bears the CFTR G542X nonsense mutation in the endogenous locus using CRISPR/Cas9 gene editing. In addition to displaying severe CF manifestations and developmental defects such as reduced growth, abnormal tooth enamel, and intestinal obstruction, CFTR G542X knockin rats demonstrated an absence of CFTR function in tracheal and intestinal sections as assessed by nasal potential difference and transepithelial short-circuit current measurements. Reduced CFTR mRNA levels in the model further suggested sensitivity to nonsense-mediated decay, a pathway elicited by the presence of PTCs that degrades the PTC-bearing transcripts and thus further diminishes the level of CFTR protein. Although functional restoration of CFTR was observed in G542X rat tracheal epithelial cells in response to single readthrough agent therapy, therapeutic efficacy was not observed in G542X knockin rats in vivo. The G542X rat model provides an invaluable tool for the identification and in vivo validation of potential therapies for CFTR nonsense mutations.
Objectives: Retrotransposable elements (REs), consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), are a group of markers that can be useful for human identity testing. Until now, however, due to the inherent size difference (up to 6 kb in some instances) associated with insertion and null alleles (or INNULs), the use of REs for facilitated population studies has not been sought or practical. The size of the insertion elements (from a few hundred to several thousand bp) has proven to limit their utility as a marker because of the inefficient amplicon yield with PCR. A novel primer design now facilitates INNUL marker testing. A preliminary panel of single-locus markers was developed to evaluate the potential of typing these insertion elements. Nine INNULs (5 Alu and 4 LINEs) were typed in three major North American populations and analyzed for population genetic features. In addition, the variation of each marker among the sample populations provides insight of its potential use as individual identification or ancestral marker. Methods: INNUL markers were developed into fluorescently labeled single-loci PCR. Nine markers were developed with amplicons that were less than 180 bp in length, and, depending on the locus amplicons of the INNULs, alleles varied in size from 50 to 1 bp. This allele size is noteworthy because the insertion alleles of the 9 loci range in size from 297 to 6,195 bp. The allele distribution of the INNULs was assessed and analyzed in three major North American populations. Results: Upon observation of the distribution of the alleles in three major North American populations, the markers generally met Hardy-Weinberg expectations, and there was little evidence of detectable levels of linkage disequilibrium. Due to varying distributions of the alleles in the major population groups tested, some of the markers might be better suited for use as an individual identification marker, while others are better suited for bio-ancestral studies. Conclusions: Using the primer design strategy described in our work, SINEs and (for the first time, to our knowledge) LINEs can be utilized as markers for studying population genetic variation that is more amenable to the limitations of the PCR technique. This study lays the foundation for future work of developing a multiplex panel of INNUL markers that can be used as a single-tube assay for human identity testing utilizing small amplicons (<180 bp), which could be useful for ancient or degraded forensic DNA samples.
ID 53194 Poster Board 252Over the last 3 years, the COVID-19 pandemic has severely affected human lives and the global economy. 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, wild type 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 novel humanized ACE2 models can be valuable for COVID-19 research.
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