Gene function & annotation

Exploring the influence of genomic variation on CRISPR/Cas9 guide RNA design in cattle

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Abstract

This study explores how genomic variation among cattle influences CRISPR/Cas9 guide RNA (gRNA) design considerations. CRISPR/Cas9 editing relies on gRNA-mediated targeting of specific genomic loci. Therefore, gRNA design is a crucial step in optimizing gene editing in livestock. To date, gRNA design has relied primarily on reference genome assemblies that represent a consensus sequence and do not capture individual genetic variation introduced by single nucleotide polymorphisms (SNPs) and insertions or deletions (indels). Human studies have shown that this non-reference genetic variation can alter gRNA target specificity, but similar research in cattle is limited. Cas nucleases require specific three-base protospacer adjacent motifs (PAMs), such as 5′-NGG-3′, immediately downstream of the target site for DNA recognition and cleavage. In this study, approximately 281 million (280,752,635) NGG motifs were identified in the Bos taurus reference genome (ARS-UCD1.3), derived from a Hereford cow, L1 Dominette 01449. Genetic variation that is not represented in the reference genome can result in the creation or destruction of PAMs. Created PAMs are loci where non-reference SNPs change NHG or NGH motifs to NGG, while destroyed PAMs are loci where non-reference SNPs alter NGG motifs to NHG or NGH. Variation in PAM presence can alter the set of genomic sites available for gRNA targeting. In addition, increased sequence similarity between intended targets and other genomic loci can influence predicted off-target profiles. To assess the effects of breed-specific genetic variation in cattle, whole genome sequencing (WGS) was performed (Revio PacBio) on three sires, each representing a distinct Bos taurus breed: Angus, Charolais, and Jersey. Additionally, a composite genome was constructed by integrating all publicly available Bos taurus variants to represent the genetic diversity present within the population (ensembl.org). Analysis found that all three sire genomes and the composite genome each contained approximately 281 million PAM sites, consistent with the reference genome (Table 1). Compared to the reference genome, fewer than 1.1% of PAM sites were altered (created or destroyed) in each sire-specific genome, while more than 12% were altered in the composite genome (Table 1). Additionally, previously published bovine gRNA sequences were compiled and benchmarked using commonly recommended mismatch-based criteria, revealing considerable variability in gRNA design practices, with nearly half (33/70) not meeting the benchmark. While human studies have recommended whole-genome sequencing as best practice for accounting for sequence variation in clinical settings, this approach is not practical or scalable for most livestock applications. Accordingly, this study highlights opportunities to further standardize gRNA design practices and to develop practical, variant-aware tools that can improve gRNA consistency and editing efficiency while remaining compatible with current livestock breeding and gene-editing workflows.

Keywords: 2026

How to Cite:

Finchum, R., Mueller, M. & Muir, W., (2026) “Exploring the influence of genomic variation on CRISPR/Cas9 guide RNA design in cattle”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2287008. doi: https://doi.org/10.31274/wcgalp.24196

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