Assessment of Mosaicism and Detection of Cryptic Alleles in CRISPR/Cas9-Engineered Neurofibromatosis Type 1 and TP53 Mutant Porcine Models Reveals Overlooked Challenges in Precision Modeling of Human Diseases.

Autor:	Rubinstein CD; Biotechnology Center, University of Wisconsin-Madison, Madison, WI, United States., McLean DT; Biotechnology Center, University of Wisconsin-Madison, Madison, WI, United States., Lehman BP; Biotechnology Center, University of Wisconsin-Madison, Madison, WI, United States., Meudt JJ; Biomedical and Genomic Research Group, Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI, United States., Schomberg DT; Biomedical and Genomic Research Group, Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI, United States., Krentz KJ; Biotechnology Center, University of Wisconsin-Madison, Madison, WI, United States., Reichert JL; Swine Research and Teaching Center, Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI, United States., Meyer MB; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, United States., Adams M; Biotechnology Center, University of Wisconsin-Madison, Madison, WI, United States., Konsitzke CM; Biotechnology Center, University of Wisconsin-Madison, Madison, WI, United States., Shanmuganayagam D; Biomedical and Genomic Research Group, Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI, United States.; Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States.
Jazyk:	angličtina
Zdroj:	Frontiers in genetics [Front Genet] 2021 Sep 23; Vol. 12, pp. 721045. Date of Electronic Publication: 2021 Sep 23 (Print Publication: 2021).
DOI:	10.3389/fgene.2021.721045
Abstrakt:	Genome editing in pigs has been made efficient, practical, and economically viable by the CRISPR/Cas9 platform, representing a promising new era in translational modeling of human disease for research and preclinical development of therapies and devices. Porcine embryo microinjection provides a universally available, efficient option over somatic-cell nuclear transfer, but requires that critical considerations be made in genotypic validation of the models that routinely go unaddressed. Accurate validation of genotypes is especially important when modeling genetic disorders, such as neurofibromatosis type 1 (NF1) that exhibits complex genotype-phenotypic relationships. NF1, an autosomal dominant disorder, is particularly hard to model as it manifests very differently across patients, and even within families, with over 3,000 disease-associated mutations of the neurofibromin 1 ( NF1 ) gene identified. The precise nature of the mutations plays a role in the complex phenotypic presentation of the disorder that includes benign and malignant peripheral and central nervous system tumors, a variety of motor deficits and debilitating cognitive impairments and musculoskeletal, cardiovascular, and gastrointestinal disorders. NF1 can also often involve mutations in passenger genes such as TP53 . In this manuscript, we describe the creation of three novel porcine models of NF1 and a model additionally harboring a mutation in TP53 by embryo microinjection of CRISPR/Cas9. We present the challenges encountered in validation of genotypes and the methodological strategies developed to counter the hurdles. We present simple options for quantifying level of mosaicism: a quantitative method (targeted amplicon sequencing) for small edits such as SNPs and indels and a semiquantitative method (competitive PCR) for large edits. Characterization of mosaicism allowed for strategic selection of founder pigs for rapid, economical expansion of genetically defined lines. We also present commonly observed unexpected DNA repair products (i.e., structural variants or cryptic alleles) that are refractory to PCR amplification and thus evade detection. We present the use of copy number variance assays to overcome hurdles in detecting cryptic alleles. The report provides a framework for genotypic validation of porcine models created by embryo microinjection and the expansion of lines in an efficient manner. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2021 Rubinstein, McLean, Lehman, Meudt, Schomberg, Krentz, Reichert, Meyer, Adams, Konsitzke and Shanmuganayagam.)
Databáze:	MEDLINE
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