RI(dl)-2

CRISPR-Cas9 HDR knock-in uses a guide RNA to direct Cas9 to a genomic target adjacent to a PAM, where Cas9 creates a double-strand break; if a donor DNA template with homology to the cut region is present, cellular HDR can copy the donor sequence into the genome, producing a precise substitution, tag, reporter, or insertion rather than an indel. The readout is the fraction of alleles or cells carrying the intended donor-derived edit, measured by junction PCR, restriction-fragment analysis, Sanger sequencing, amplicon deep sequencing, flow cytometry for reporter knock-in, or clone genotyping; NHEJ indels and partial or non-HDR insertions are measured in parallel because they compete with or confound precise HDR outcomes.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.