JAX_DPC: Protocol to generate whole-gene knockout (KO) and critical exon (CE) deletion in iPSCs (KOLF2.2) v1

• DOI: 10.17504/protocols.io.e6nvwb24wvmk/v1
• Publication Date: 2025-01-31T08:59:49Z
• Authors: Enrica Pellegrino; JUSTIN.MCDONOUGH ...; William C. Skarnes
• Keywords: Guide RNA; Gene knockin; Gene knockout; Coding region

Precise deletions are performed in human induced pluripotent stem cells (iPSC) by Cas9 RNP-mediated homology directed repair with oligonucleotide templates that span the desired breakpoints. We aimed to delete 1) a ‘critical exon’ (CE) common all transcript isoforms that, when deleted, causes frameshift coding sequence or 2) up 100 kb of containing most protein (whole-gene knockout, KO). A bridging oligo two degenerate nucleotides at breakpoint was designed promote HDR and barcoding precise deletions. pair ‘PAM out’ guide RNAs low off-target scores calculated WGE were selected define endpoints deletion. Using nucleofection, we co-deliver synthetic harboring degenerative bases deletion break point along recombinant single RNA (sgRNA) target specific gene interest.This protocol ensures efficient editing generation loss-of-function alleles both copies gene.The allows us rule out on-target effects such as loss one copy identify independent, non-sibling clones.