CRISPR troubleshooting in non-model organism: delivery, specificity, validation

[George87]

As a graduate student in molecular biology, I'm designing my thesis project around using CRISPR gene editing to investigate a specific gene's function in a non-model organism, which presents unique challenges compared to standard lab mice or cell lines. While I'm confident in the core technique, I'm concerned about optimizing delivery methods and ensuring specificity to avoid off-target effects that could invalidate my results. For researchers with hands-on CRISPR experience in complex systems, what were your most valuable troubleshooting steps when you first established your protocol, and how did you rigorously validate your edits and confirm phenotypic changes were due to your target gene and not collateral damage? I'm particularly interested in practical advice on gRNA design and validation assays beyond just sequencing.