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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #409080
Research Project: Validate Causative Mutations in Agriculturally-Important Vertebrates

Location: Plant Genetics Research

Title: Novel off-targeting events identified after genome wide analysis of CRISPR-Cas edited pigs

Author
item Redel, Bethany
item YOON, JUNCHUL - University Of Missouri
item REESE, EMILY - University Of Missouri
item AN, HONG - University Of Missouri
item UH, KYUNGJUN - University Of Missouri
item Chen, Paula
item PRATHER, RANDALL - University Of Missouri
item LEE, KIHO - University Of Missouri

Submitted to: The CRISPR Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/22/2024
Publication Date: 6/21/2024
Citation: Redel, B.K., Yoon, J., Reese, E., An, H., Uh, K., Chen, P.R., Prather, R.S., Lee, K. 2024. Novel off-targeting events identified after genome wide analysis of CRISPR-Cas edited pigs. The CRISPR Journal. 7(3): 141 - 149. https://doi.org/10.1089/crispr.2024.0012.
DOI: https://doi.org/10.1089/crispr.2024.0012

Interpretive Summary: Genome editing technology has revolutionized our ability to create genetic modifications in cells and animals. The main concern with using this technology is the potential to produce unknown and unwanted modifications, known as off-targeting, elsewhere in the genome of these animals and/or cells. These unintended modifications may have a negative impact on the animal phenotype. Because of this concern, it is important that we 1) are able to identify off-targeting in the genome of animals or cells whose genome has been edited, and 2) identify ways to diminish these effects. Here, we illustrate a pipeline that can detect previously unknown off-targeting events by using whole genome sequencing and off-target prediction software to filter locations in the genome where true off-targeting can occur in pigs. This will be important to accelerate the development of useful genome edited animals.

Technical Abstract: CRISPR-Cas technology has transformed our ability to introduce targeted modifications, allowing unconventional animal models such as pigs to model human diseases and improve its value for food production. The main concern with using the technology is the possibility of introducing unwanted modifications in the genome. In this study, we illustrate a pipeline to comprehensively identify off-targeting events on a global scale in the genome of three different gene-edited pig models. Whole genome sequencing paired with an off-targeting prediction software tool filtered off-targeting events amongst natural variations present in gene-edited pigs. This pipeline confirmed two known off-targeting events in IGH knockout pigs, AR and RBFOX1, and identified other presumably off-targeted loci. Independent validation of the off-targeting events using other gene-edited DNA confirmed two novel off-targeting events in RAG2/IL2RG knockout pig models. This unique strategy offers a novel tool to detect off-targeting events in genetically heterogeneous species after genome editing.