JAX_DPC: Protocol for generating PTC+1 knockout alleles in iPSCs (KOLF2.2J) v1

• DOI: 10.17504/protocols.io.5qpvo92dxv4o/v1
• Publication Date: 2025-01-31T09:00:17Z
• Authors: Enrica Pellegrino; JUSTIN.MCDONOUGH ...; William C. Skarnes
• Keywords: Gene knockout; Gene knockin; Conditional gene knockout

The ‘PTC+1’ knockout strategy offers a robust and efficient approach to eliminate gene function in human induced pluripotent stem cells (iPSCs). method utilizes precise genome engineering introduce premature termination codon (PTC) degenerate base insertion (+1) within an early coding exon that is shared across all transcript isoforms. Here, we describe the protocol for generation of alleles KOLF2.2J iPSCs achieve targeted knockout. Using nucleofection, co-deliver synthetic oligonucleotide harboring PTC+1 mutation along with recombinant Cas9 protein single guide RNA (sgRNA) designed target specific interest.This ensures editing loss-of-function both copies gene.The allows us rule out on-target effects such as loss one copy identify independent, non-sibling clones.