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

Abstract

CRISPR-Cas9 editing in primary bone marrow-derived macrophages (BMD-M{phi}s) requires coordinated assessment of cellular morphology, target modification, and sequence-level editing outcomes. We describe a workflow for dual-guide targeting of murine Pla2g6 that integrates guide design, ribonucleoprotein nucleofection, PCR-heteroduplex mobility analysis (PCR-HMA), and targeted amplicon sequencing. At 24 h after nucleofection, cells receiving ribonucleoproteins assembled with chemically synthesized single-guide RNAs preserved the elongated, adherent morphology, whereas cultures receiving in vitro-transcribed guides contained predominantly poorly adhered, rounded cells and appeared less densely populated; both guide preparations generated indels and deletions, so the difference observed was in cell appearance rather than in target modification. PCR-HMA provided a simple, qualitative screen for sequence heterogeneity and for a putative deletion product, which was subsequently characterized by sequencing. In two independent BMDM samples, one each from C57BL/6 and NOD mice, breakpoint-based analysis identified editing-associated indels in 54.7% and 44.5% of analyzed reads, compared with 0.28% and 0.07% in matched Cas9-only controls. Deletions spanning both target sites represented 33.1% and 26.2% of analyzed reads; none were detected among 4,383 analyzed control reads. The modal spanning deletion was 137 bp, the size predicted from precise joining of the two blunt Cas9 ends. Sequence interpretation depended on verified guide coordinates, deletion-aware analysis settings, and matched controls: scored permissively, the same control reads returned 22-23% apparent editing. Modest-depth Nanopore sequencing recovered the major editing classes, although low-frequency variants and small indels required additional caution. This workflow supports a simple and streamlined screening and sequence-based genotyping of edited BMDM populations, allowing the discernment of the comprehensive mutation spectrum arising from CRISPR-Cas9 gene targeting.