RNP-Mediated CRISPR-Cas9 Knockout of HPRT1 in HEK293T Cells by Nucleofection

Three independent nucleofection replicates per condition performed on separate days. Conditions: (1) HPRT1-targeting RNP, (2) non-targeting scrambled sgRNA RNP, (3) Cas9-only (no sgRNA), (4) mock (buffer only). Each replicate uses 2 x 10^5 cells. Bulk indel readout at 72 h post-nucleofection (T72) before selection; 6-TG selection applied at day 4 for 10 days; 24 clones per targeting replicate picked and genotyped. Sample size of 24 clones gives >99% probability of recovering at least one biallelic frameshift clone at an expected 60% per-allele frameshift rate.

BSL-2 for HEK293T culture; work in a Class II biosafety cabinet. 6-thioguanine is a cytotoxic/mutagenic antimetabolite — handle with nitrile gloves, lab coat, and eye protection; collect 6-TG waste as chemotherapeutic/hazardous chemical waste. Cas9 RNP itself is non-hazardous. Decontaminate cultureware with 10% bleach (20 min) before disposal as biohazard. NaOH stock for 6-TG is corrosive; prepare in a fume hood.

Negative controls: non-targeting scrambled sgRNA RNP (controls for nucleofection toxicity and 6-TG background), Cas9-only RNP (controls for nuclease without guide), and mock buffer-only (baseline viability). Positive control for the selection: parental HEK293T should be killed by 6-TG (>99% death by day 10), confirming selection stringency. Reversion control: HPRT1-KO clones grown in HAT medium should die (HPRT required for the salvage pathway), confirming a true functional knockout rather than a 6-TG transporter mutation. Editing positive control: a previously validated HPRT1 sgRNA from a prior run run in parallel.

Bulk indel frequency by ICE/TIDE should be 70-90% for the HPRT1 RNP and <2% for all controls. 6-TG selection should leave <0.1% surviving colonies in controls and 5-30% in the targeted population. Among picked clones, >90% should carry frameshift indels and lack detectable HPRT protein on Western blot (expected band ~24.6 kDa absent). HAT medium should kill all confirmed KO clones within 7 days.

• HEK293T cells (ATCC CRL-3216), passage <20, cultured in DMEM high glucose + 10% FBS + 1% penicillin/streptomycin
• S. pyogenes Cas9 nuclease, NLS-tagged, 10 mg/mL (e.g., IDT Alt-R HiFi Cas9 V3 or equivalent)
• Synthetic sgRNA targeting HPRT1 exon 3 (protospacer 5'-AAGGGGGGCTATAAGTTCTT-3', PAM AGG), chemically modified (2'-O-methyl + phosphorothioate at terminal 3 nt), 100 µM stock
• Non-targeting control sgRNA (scrambled, no human genome match)
• Nucleofection kit (Lonza SF Cell Line 4D-Nucleofector X Kit) with supplemented SF solution
• 6-Thioguanine (Sigma A4882), 10 mg/mL stock in 0.1 N NaOH, used at 10 µg/mL
• HAT supplement (Gibco 21060017) for reversion control
• QuickExtract DNA Extraction Solution (Lucigen)
• HPRT1 genotyping primers (F 5'-CTGGATTACATCAAAGCACTG-3', R 5'-TGGCTTATATCCAACACTTCG-3'), amplicon ~600 bp

Disrupt the X-linked HPRT1 locus in HEK293T cells using pre-assembled Cas9 RNP delivered by nucleofection, then isolate and validate clonal knockout lines. HPRT1 is chosen because functional loss confers resistance to 6-thioguanine (6-TG), providing a phenotypic enrichment that orthogonally confirms editing efficiency at both bulk and clonal levels.

1. Seed HEK293T at 3 x 10^5 cells/well in a 6-well plate 24 h before nucleofection; harvest at 70-80% confluence.
2. Assemble RNP: combine 104 pmol Cas9 with 120 pmol sgRNA (1:1.15 molar) in SF solution to 5 µL total; incubate 15 min at room temperature.
3. Harvest cells, count, pellet 2 x 10^5 cells at 200 x g for 5 min, aspirate fully.
4. Resuspend pellet in 20 µL supplemented SF solution; add the 5 µL RNP; mix gently.
5. Transfer to a 16-well Nucleocuvette strip; pulse program CM-130 on the Lonza 4D-Nucleofector.
6. Add 80 µL pre-warmed antibiotic-free DMEM/10% FBS; rest 10 min at 37 C.
7. Transfer to one well of a 24-well plate with 0.5 mL complete medium; incubate 37 C, 5% CO2.
8. At T72, harvest 30% of cells for genomic DNA via QuickExtract (65 C 6 min, 98 C 2 min) for indel quantification.
9. At day 4, replace medium with complete medium + 10 µg/mL 6-TG; refresh every 3 days for 10 days.
10. Single-cell sort or limiting-dilute survivors into 96-well plates; expand 14-21 days.
11. Genotype clones by amplicon PCR + Sanger; confirm protein loss by Western blot.

Independent variable: presence/identity of the HPRT1-targeting sgRNA in the RNP. Dependent variables: bulk indel frequency (%), fraction of 6-TG-resistant colonies, fraction of clones that are biallelic frameshift, HPRT protein level (Western densitometry). Controlled variables: cell passage number (<20), cell count per nucleofection (2 x 10^5), RNP molar ratio (1:1.15), nucleofection program (CM-130), 6-TG concentration (10 µg/mL), selection duration (10 days), incubation temperature/CO2.

Nucleofection of a single high-activity sgRNA-Cas9 RNP targeting HPRT1 exon 3 will produce >70% indel frequency in the bulk population, and 6-TG selection will enrich frameshift biallelic (functionally hemizygous, given X-linkage in this near-triploid line) knockouts to >90% of surviving clones, with loss of HPRT enzymatic activity confirmed by 6-TG resistance and Western blot.

Quantify bulk indels from Sanger chromatograms using ICE (Synthego) or TIDE, reporting indel % and the frameshift fraction (R^2 > 0.9 required). Clonal genotypes resolved by aligning Sanger traces to reference (e.g., Benchling/SnapGene) and, where ambiguous, by TOPO cloning and sequencing 8 colonies per clone. Western blot bands quantified in ImageJ (Fiji), normalizing HPRT signal to GAPDH loading control and expressing each clone relative to parental. Editing-efficiency comparisons summarized as mean +/- SD across the three replicates.

Low bulk editing (<30%): re-check RNP assembly molar ratio and confirm sgRNA integrity by gel; try program EH-100 or DS-150 and re-titrate Cas9 to 120 pmol. High nucleofection death in all conditions: cells over-confluent or wrong solution — use SF (not SE/SG) for HEK293T and harvest at <80%. No 6-TG-resistant colonies despite good bulk editing: 6-TG lot inactive or concentration off — verify parental cells are killed and re-prepare stock. Many 6-TG-resistant clones are wild-type at HPRT1: spontaneous transporter mutations — confirm with HAT reversion and re-pick. Mixed/heterozygous Sanger traces in clones: subclone again by single-cell sorting to ensure clonality.

Compare bulk indel frequency between targeting and control conditions with a one-way ANOVA across the four conditions (n = 3 independent nucleofections) followed by Dunnett's post hoc test versus mock, alpha = 0.05. Western densitometry (KO clones vs parental) compared by unpaired two-tailed t-test. With n = 3 and an expected effect size of >5 SD between targeting and control indel rates, power exceeds 0.95. Report exact p-values; correct for the multiple post-hoc comparisons via Dunnett's built-in adjustment.