A multi-omics and population genetics framework identifies deployable genomic safe harbors for CRISPR knock-in in the Pacific oyster (Crassostrea gigas)

Precise CRISPR knock-in can install reporters, regulatory elements, or favorable alleles for trait engineering in aquaculture species. In mollusks, this remains difficult in practice because there are few validated genomic safe harbors (GSHs) that allow reliable, long-term expression without disturbing nearby gene regulation. Here we developed an oyster-specific workflow to nominate and prioritize GSH candidates for targeted knock-in in Crassostrea gigas by combining genome annotation with multi-omics filters and practical design constraints. First, we adapted the screening rules to oyster genome architecture by setting buffers around coding genes and requiring sufficiently long continuous intergenic segments, and then applied lncRNA, ATAC-seq, and DNA methylation filters to nominate 21 engineering-grade candidates. Second, we compared these 21 candidates with tiered, length-matched control regions and found that they are consistently located in quiet genomic neighborhoods: fewer nearby coding genes, little overlap with union regulatory peaks, and low intergenic RNA-seq signal. Third, population resequencing showed fewer variants in 21 candidate regions, which improves the chances that the same sgRNA targets and homology arms can be reused across individuals. Cross-species comparisons, however, indicated limited collinearity beyond the closest sister lineage. Finally, we used a transparent multi-criteria decision analysis (MCDA) score that integrates insulation, regulatory inactivity, genetic context, and CRISPR designability to generate a stable ranking of loci. This work provides an oyster GSH genomic resource and a practical workflow, offering an important reference for future trait-oriented knock-in studies in oysters.