How to troubleshoot CRISPR efficiency reagents vs nucleofection in mammalian cells

[ScarlettT]

I'm a molecular biology PhD student, and my research involves using CRISPR gene editing to create specific knockouts in a mammalian cell line. I'm struggling with consistently low editing efficiency despite optimizing guide RNA design and delivery protocols. For others working with similar systems, what troubleshooting steps have you found most critical? I'm particularly interested in your experiences with different transfection reagents versus nucleofection, and how you verify successful editing beyond just PCR and Sanger sequencing—are there reliable functional assays you'd recommend? How do you manage off-target effects in your experimental design, and what controls are absolutely non-negotiable in your lab?