An integrated workflow for dual-guide CRISPR-Cas9 targeting and amplicon-based genotyping of Pla2g6 in primary mouse bone marrow-derived macrophages

Dual-guide CRISPR-Cas9 targeting of murine Pla2g6 in primary BMD-Mϕs yields deletion-spanning edits in 33.1% and 26.2% of reads, far above Cas9-only controls.

• Why it matters: Editing in primary macrophages needs coordinated, sequence-validated genotyping alongside checks that cells remain suitable for downstream assays.
• What they did: The workflow combines guide design, ribonucleoprotein nucleofection, PCR-HMA at 24 h, and targeted amplicon sequencing, using C57BL/6 and NOD BMDM samples with matched Cas9-only controls.
• The result: Editing-associated indels were found in 54.7% and 44.5% of analyzed reads versus 0.28% and 0.07% in controls, enabling mutation-spectrum screening and deletion-aware interpretation.

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