Problem	Potential solution
Failed ICE analysis (step A4)	Try to improve the PCR, the sequencing quality, or both. A clearly defined PCR band and high-quality sequencing data are required for successful ICE analysis.
Low gene knock-out efficiency (step A4)	Efficient gRNAs are required: Try different gRNAs. Please test several gRNAs to identify highly efficient ones.
Inefficient AAV production/low AAV titers (section E)	Use single-stranded DNA as a donor template if the size of the donor template is close to 4.4 kb.
Inefficient expansion of T cells after gene editing (step F18/F19)	Expand cells gradually from a 96-well U-bottom plate to a bigger plate (48-, 24-, 12-, and 6-well plates) and monitor the culture every day until cells have sufficiently expanded to be used in downstream analysis or applications.