CRISPR-Cas9 gene knockout relies on a guide RNA directing the Cas9 nuclease to a specific 20-base-pair genomic sequence adjacent to a PAM site, where Cas9 introduces a double-strand break that the cell resolves through one of two repair pathways: homology-directed repair (HDR), which uses a donor template with long homology arms to precisely insert a designed sequence, or non-homologous end joining (NHEJ), an error-prone pathway that produces random insertions/deletions disrupting the reading frame. In diploid organisms, biallelic (double) gene knockout requires both chromosomal copies (alleles) of a gene to be disrupted, which can occur through combinations of donor-mediated insertion and indel formation across the two alleles, situating this within the molecular biology domain of genome editing and gene function analysis.
CRISPR-Cas9 gene knockout relies on a guide RNA directing the Cas9 nuclease to a specific 20-base-pair genomic sequence adjacent to a PAM site, where Cas9 introduces a double-strand break that the ce…