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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

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 univ...

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Autores principales: Rubinstein, Clifford Dustin, McLean, Dalton T., Lehman, Brent P., Meudt, Jennifer J., Schomberg, Dominic T., Krentz, Kathy J., Reichert, Jamie L., Meyer, Mark B., Adams, Marie, Konsitzke, Charles M., Shanmuganayagam, Dhanansayan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495252/
https://www.ncbi.nlm.nih.gov/pubmed/34630515
http://dx.doi.org/10.3389/fgene.2021.721045
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author Rubinstein, Clifford Dustin
McLean, Dalton T.
Lehman, Brent P.
Meudt, Jennifer J.
Schomberg, Dominic T.
Krentz, Kathy J.
Reichert, Jamie L.
Meyer, Mark B.
Adams, Marie
Konsitzke, Charles M.
Shanmuganayagam, Dhanansayan
author_facet Rubinstein, Clifford Dustin
McLean, Dalton T.
Lehman, Brent P.
Meudt, Jennifer J.
Schomberg, Dominic T.
Krentz, Kathy J.
Reichert, Jamie L.
Meyer, Mark B.
Adams, Marie
Konsitzke, Charles M.
Shanmuganayagam, Dhanansayan
author_sort Rubinstein, Clifford Dustin
collection PubMed
description 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.
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spelling pubmed-84952522021-10-08 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 Rubinstein, Clifford Dustin McLean, Dalton T. Lehman, Brent P. Meudt, Jennifer J. Schomberg, Dominic T. Krentz, Kathy J. Reichert, Jamie L. Meyer, Mark B. Adams, Marie Konsitzke, Charles M. Shanmuganayagam, Dhanansayan Front Genet Genetics 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. Frontiers Media S.A. 2021-09-23 /pmc/articles/PMC8495252/ /pubmed/34630515 http://dx.doi.org/10.3389/fgene.2021.721045 Text en Copyright © 2021 Rubinstein, McLean, Lehman, Meudt, Schomberg, Krentz, Reichert, Meyer, Adams, Konsitzke and Shanmuganayagam. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Rubinstein, Clifford Dustin
McLean, Dalton T.
Lehman, Brent P.
Meudt, Jennifer J.
Schomberg, Dominic T.
Krentz, Kathy J.
Reichert, Jamie L.
Meyer, Mark B.
Adams, Marie
Konsitzke, Charles M.
Shanmuganayagam, Dhanansayan
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495252/
https://www.ncbi.nlm.nih.gov/pubmed/34630515
http://dx.doi.org/10.3389/fgene.2021.721045
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