CRISPR-Cas9 Ribonucleoprotein Knock-In of a GFP Reporter at the Endogenous SOX2 Locus in Human iPSCs via HDR with ssODN/Plasmid Donor

One iPSC line is electroporated with RNP + donor in triplicate cuvettes; a no-donor (RNP-only) arm measures cutting (indel) efficiency, and a no-RNP (donor-only) arm controls for random integration. After recovery, single cells are sorted into 96-well plates; ~96-192 clones are screened by junction PCR. Targeted clones (target n >= 6) are expanded, sequence-verified at both junctions, tested for off-target editing at top predicted sites, and karyotyped. GFP fidelity is validated against SOX2 antibody co-staining.

BSL-1/BSL-2 for iPSC and recombinant-DNA work; institutional IBC approval required for CRISPR editing of human cells. Handle in a Class II cabinet. Cas9 protein and synthetic guides are non-hazardous but treat donor plasmids per recombinant-DNA SOP. Electroporation buffers and cuvettes are single-use; dispose as biohazard. Ethidium bromide/gel stains and sequencing reagents per SOP. Decontaminate cells and media with 10% bleach. No viral vectors are used (RNP delivery), reducing biosafety burden. Gloves, coat, eye protection; sharps segregated.

RNP-only (no donor) control quantifies cutting/indel efficiency and provides an NHEJ reference. Donor-only (no RNP) control measures random/illegitimate integration background. Untransfected wild-type cells control for autofluorescence in the GFP gate and provide a SOX2-staining baseline. A non-targeting sgRNA RNP controls for electroporation toxicity. Junction PCR includes a wild-type genomic positive control and a no-template negative control. Off-target sites compared against the same loci in the parental line.

Bulk cutting >50% indels by ICE/TIDE in the RNP-only arm. HDR knock-in detectable in >=5% of screened clones (often 2-15%). Correctly targeted clones show clean 5' and 3' junction PCR bands and in-frame insert by Sanger. GFP fluorescence in pluripotent cells and strong GFP up in neural progenitors, concordant with SOX2 antibody (>90% overlap). Top off-target sites show no significant indels above parental background. KaryoStat normal in final clones.

• Human iPSC line (karyotype-normal, p25-p40)
• Recombinant Cas9 protein (HiFi/SpCas9), NLS-tagged
• Chemically modified synthetic sgRNA targeting SOX2 3' end (near stop codon)
• HDR donor: plasmid with P2A-GFP + 500-800 bp homology arms, or symmetric ssODN (for short tags)
• Nucleofection system + P3 Primary Cell kit (4D-Nucleofector) or Neon kit
• mTeSR Plus + 10 µM Y-27632; CloneR or equivalent single-cell cloning supplement
• HDR enhancer (e.g., a NHEJ-pathway inhibitor / SCR7 alternative)
• Vitronectin coating; Accutase
• Genotyping: high-fidelity polymerase, junction PCR primers (one in genome outside arm, one in insert), Sanger sequencing
• Mismatch/indel assay (T7E1 or ICE/TIDE via Sanger)
• KaryoStat or G-banding; anti-SOX2 antibody for GFP validation

To insert an in-frame GFP (or P2A-GFP) cassette at the endogenous SOX2 stop codon in human iPSCs using Cas9 ribonucleoprotein (RNP) delivery and homology-directed repair (HDR) with a donor template, then isolate and validate clonal lines that are heterozygous or homozygous knock-in, off-target-clean, and genomically stable for use as a live SOX2 reporter.

1. Assemble RNP: incubate Cas9 (100-120 pmol) + sgRNA (120-150 pmol) 10-15 min at RT.
2. Dissociate iPSCs (Accutase), resuspend 1-2 x 10^5 cells in P3 nucleofection solution; add RNP + donor (1-2 µg plasmid or 100 pmol ssODN).
3. Electroporate (4D-Nucleofector program CB-150 or optimized); immediately add warm mTeSR Plus + Y-27632 + HDR enhancer; plate on vitronectin.
4. 24-48 h: replace with mTeSR Plus (remove enhancer); recover 3-5 days.
5. Measure bulk cutting: extract DNA from the RNP-only arm; run T7E1 or ICE/TIDE on the SOX2 amplicon.
6. Single-cell clone: dissociate, sort 1 cell/well into vitronectin-coated 96-well plates in CloneR; grow 10-12 days.
7. Screen clones by junction PCR (5' and 3'); Sanger-sequence positive junctions; confirm zygosity.
8. Validate: differentiate select clones, co-stain GFP vs. SOX2; assess top off-target sites by amplicon sequencing; karyotype.

Independent variables: donor format (plasmid vs. ssODN), HDR-enhancer presence, RNP:donor ratio. Dependent variables: indel/cutting efficiency (%), HDR/knock-in rate (% targeted clones), clone survival, off-target indel frequency, GFP-SOX2 concordance, karyotype status. Controlled variables: cell number per nucleofection, Cas9:sgRNA molar ratio, nucleofection program, recovery medium, single-cell cloning supplement, homology-arm length.

Electroporation of a pre-assembled Cas9-sgRNA RNP targeting the SOX2 3' region, together with an HDR donor bearing ~500-800 bp homology arms (plasmid) or symmetric ssODN, plus an HDR-enhancing small molecule, will achieve >5% knock-in among surviving cells. Single-cell cloning and junction PCR/sequencing will recover correctly targeted clones whose GFP faithfully reports endogenous SOX2 expression in pluripotent cells and neural progenitors.

Indel efficiency computed by ICE or TIDE from Sanger traces (RNP-only vs. wild-type). Junction PCRs scored for correct band size; Sanger reads aligned in SnapGene/Benchling to confirm scarless in-frame insertion and zygosity. Off-target amplicon-seq analyzed by CRISPResso2 for indel frequencies at predicted sites. GFP-SOX2 concordance quantified in Fiji as percent GFP+ nuclei that are SOX2+. KaryoStat .cel files analyzed in ChAS for copy-number integrity.

1. Low cutting efficiency: sgRNA poorly designed or degraded — redesign near the cut site, use chemically modified guides, and re-titrate RNP. 2. High toxicity/low survival post-nucleofection: too many cells or harsh program — reduce to 1 x 10^5 cells, add Y-27632, and try a gentler 4D program. 3. No HDR clones: enhance HDR (cold-shock 32 C 24-48 h, add NHEJ inhibitor) and lengthen homology arms to 800 bp. 4. Only random integration (donor-only signal): use ssODN or linearized donor and include a 5' junction primer outside the arm to confirm site-specific insertion. 5. GFP not reporting SOX2 faithfully: confirm in-frame fusion/P2A and that the tag did not disrupt the SOX2 ORF; re-sequence the junction.

HDR rate compared between donor formats by Fisher's exact test on targeted/total clones (alpha = 0.05). Off-target indel frequencies in edited clones vs. parental compared per site by two-proportion z-test with Benjamini-Hochberg FDR correction across all tested off-target loci. n = 3 independent electroporations for bulk efficiency; clone-level n >= 96 screened. GFP-SOX2 concordance reported as mean +/- SD across >=5 fields. Power: 96 clones detects a 5% HDR rate with >95% probability of recovering >=1 positive.