# TP53 C-terminal mCherry Knock-in -- Design Report

**AI Bioinformatician by Labterna**  |  29 June 2026  |  GRCh38/hg38  |  TP53 NM_000546.6

> **Design delivered; one biology caveat to read first.** A complete, cross-validated SpCas9 + HDR design for endogenous C-terminal mCherry tagging of TP53 is provided: a primary and backup guide at the stop codon (validated across three independent off-target tools, **no pseudogene/p53-family off-targets**), an 800/800 bp HDR donor with a codon-optimized BsaI/BsmBI-free mCherry and built-in re-cut protection, and a full genotyping primer set. **Caveat:** HEK293T expresses SV40 large T antigen, which binds and functionally inactivates p53 -- p53 dynamics under genotoxic stress in this line are non-physiological (Section 7).

## 1. Objective

Design an endogenous C-terminal mCherry knock-in at TP53 (NM_000546.6, GRCh38) in HEK293T for live-cell imaging of p53 under genotoxic stress, using SpCas9 and an HDR donor. Deliverables requested: (i) guide(s) at the TP53 stop codon cross-validated across >=2 tools (a previous design failed on an undetected pseudogene off-target); (ii) an HDR donor with ~800 bp homology arms, a GS linker before mCherry, and PAM-blocking silent mutations; (iii) junction genotyping primers (5' and 3', anchored outside the arms) plus an internal Sanger pair across the insert; (iv) a full off-target report for the top guides. mCherry to be human codon-optimized with no internal BsaI/BsmBI sites.

## 2. Target biology and tagging strategy

TP53 sits on chr17p13.1 on the **minus strand**; NM_000546.6 encodes the canonical 393-aa p53 (isoform a). The C-terminus (...Lys-Leu-Met-Phe-Lys-Thr-Glu-Gly-Pro-Asp-Ser-Asp393) lies in the last exon (exon 11), and the entire 1,188-nt 3'UTR is intronless and contiguous with the stop codon in the genome -- so both homology arms can be taken directly from genomic sequence with no intron complications. mCherry is inserted **in-frame immediately before the native TGA stop** (chr17:7669609-7669611), so the fusion reuses the endogenous stop codon and 3'UTR, preserving native post-transcriptional regulation. The resulting ORF is p53(1-393)-(GGGGS)x3-mCherry-stop.

**Why C-terminal, and the caveat it carries.** The N-terminus carries the transactivation domains and the MDM2-binding site (residues ~17-29); an N-terminal tag would block MDM2 binding and artificially stabilize p53. The C-terminus is therefore the conventional choice, but it abuts the **C-terminal regulatory domain (CTD, ~363-393)** -- the site of MDM2-mediated ubiquitination (Lys370/372/373/381/382/386) and numerous regulatory PTMs (Kruse & Gu 2009). The tag is fused after residue 393 (the CTD lysines are preserved), and a flexible (GGGGS)x3 linker is used to minimize steric interference, but tagged-p53 function must be validated experimentally (Section 7).

Endogenous fluorescent tagging of p53 for live single-cell dynamics is well precedented (the p53-Mdm2 pulsing literature, Lahav et al. 2004 [9], and later endogenous reporter lines), which motivates tagging at the native locus rather than overexpressing. The guide+donor set here is a de novo design for that purpose; a formal parameter-by-parameter comparison against a specific published TP53 knock-in line is noted as a possible extension (Section 7).

## 3. Guide RNA design and selection

All NGG protospacers with a predicted blunt cut within 35 bp of the insertion point were enumerated on both strands; 7 candidates were scored. On-target efficiency and specificity were obtained from **CRISPOR** (Doench-2016, MIT and CFD specificity) and genome-wide off-targets independently from **Cas-OFFinder** (Section 4). Two guides are recommended:

| Guide (role) | Spacer 5'->3' | PAM | Strand | Cut vs insert (bp) | GC% | MIT spec | CFD spec | Doench'16 |
|---|---|---|---|---|---|---|---|---|
| TP53ct_g3 (PRIMARY) | GGAGAATGTCAGTCTGAGTC | AGG | - | -6 | 50 | 70 | 87 | 49 |
| TP53ct_g2 (backup) | AACTCATGTTCAAGACAGAA | GGG | + | -18 | 35 | 55 | 73 | 63 |

**g3 (primary)** cuts just 6 bp from the insertion point (high HDR efficiency), is 50% GC, begins with G (U6 transcription-ready), and has the **highest MIT (70) and CFD (87) specificity of any guide at the stop codon**. Critically, its protospacer straddles the insertion point, so the inserted cassette splits the target -- the edited allele is no longer a Cas9 substrate, requiring no PAM-blocking mutation. Its on-target efficiency is moderate (Doench'16 49); it was chosen for specificity, cut-to-insert proximity, and intrinsic re-cut immunity rather than peak predicted cutting.

**On the backup.** For a single-point knock-in both guides must sit at the stop codon to drive HDR there, so the standard "backup amplicon in a different region, >=100 bp away" rule does not apply -- g3 and g2 necessarily overlap (cuts ~12 bp apart). g2's genuine value as a backup is its **different seed, strand and PAM**: it is immune to a SNP under g3's seed or to g3-specific inefficiency, and it has the higher predicted efficiency (Doench'16 63), so it is the natural fallback if g3 cutting proves weak in validation. It does **not** protect against a locus-level failure (a deletion/structural variant spanning the stop codon would disable both); that residual shared-failure mode is inherent to point knock-ins. Because g2's target lies entirely within the 5' arm, a silent PAM-block is built into the donor (Section 5). Guides g4-g7 scored lower (MIT 30-61) and the GC-rich g5-g7 are markedly promiscuous (622-908 off-target sites); they are tabulated in the workbook but not recommended.

## 4. Off-target / specificity analysis (three independent tools)

Because the client's prior design failed on an off-target pseudogene match missed by a single tool, specificity was assessed with **three independent genome-wide methods** and the results cross-checked:

- **Cas-OFFinder** (local, exhaustive enumeration against hg38): NGG up to 4 mismatches plus NAG up to 3 mismatches; every hit annotated to RefSeq genes (exon/intron/intergenic).

- **CRISPOR** (web, BWA-based, hg38): independent off-target search with CFD/MIT off-target scoring and gene annotation.

- **FlashFry** (local, against a purpose-built hg38 SpCas9-NGG database): a third, independent algorithm reporting the same community metrics (CFD, Hsu/MIT, Doench).

For the primary guide **g3**, all three tools agree: **202 (Cas-OFFinder) / 184 (CRISPOR) / 176 (FlashFry) off-target sites, with zero at <=1 mismatch in every tool**. The few 2-mismatch sites are intronic/intergenic; all exonic off-targets occur only at 4 mismatches with multiple seed mismatches (max CFD 0.173, predicted inactive), and the same exonic loci (UBR1, FGA, NAALADL2) are recovered by the annotating tools. CRISPOR and FlashFry independently identify the **same single highest-risk off-target with an identical CFD of 0.612** (g3) / 0.65 (g2). **No off-target falls in a TP53 pseudogene or any p53-family gene (TP63/TP73)** -- the specific failure mode from the previous design is excluded. g2 shows the same clean pattern. Guide-level specificity is concordant: FlashFry Hsu/MIT 74 (g3) vs CRISPOR MIT 70.

| Tool (hg38) | g3 off-targets | g3 <=1 mm | g3 top-OT CFD | g2 off-targets | g2 <=1 mm | p53-family / pseudogene |
|---|---|---|---|---|---|---|
| Cas-OFFinder (local) | 202 | 0 | n/a | 299 | 0 | 0 |
| CRISPOR (web) | 184 | 0 | 0.612 | 289 | 0 | 0 |
| FlashFry (local) | 176 | 0 | 0.612 | 262 | 0 | 0* |

*FlashFry confirms the off-target count and mismatch distribution on a third algorithm; gene/pseudogene annotation is from the two annotating tools (Cas-OFFinder, CRISPOR), which agree on zero. Cas-OFFinder does not compute CFD (it enumerates exhaustively); top-OT CFD is from CRISPOR/FlashFry, which match exactly. (FlashFry's table value is genomic off-target sites (otCount); Figure 2B's bars count unique off-target sequences, slightly fewer because some map to several loci.)

**Binding-site variation.** A common SNP in a spacer seed or PAM would cause allele-specific cutting failure -- relevant here because the edit is in HEK293T's own genome. Screening Ensembl Variation (GRCh38) across the g3 and g2 protospacer+PAM windows returned 69 cataloged variants, **none common (MAF >= 1%)** under either guide -- no allele-dropout risk (g3 PASS, g2 PASS). The region is densely catalogued with rare single-submission variants, none of which threaten the assay.

## 5. HDR donor design

The donor is a **2353 bp dsDNA** (plasmid or synthetic long dsDNA) with the architecture 5' arm (800 bp) - (GGGGS)x3 linker - mCherry - 3' arm (800 bp):

- **Homology arms (~800 bp each), genomic.** 5' arm chr17:7669612-7670411 (minus strand, 5' arm); 3' arm chr17:7668812-7669611 (minus strand, 3' arm). The 5' arm ends at the last p53 sense codon (Asp393); the 3' arm begins with the native TGA stop and 3'UTR.

- **Linker.** Flexible (GGGGS)x3 (15 aa), codon-diversified to avoid a tandem repeat that would complicate synthesis/recombination.

- **mCherry.** 236-aa mCherry (Shaner 2004; GenBank AY678264, translation verified byte-identical), human codon-optimized to 708 nt (GC 61.9%), **0 internal BsaI and 0 BsmBI sites** as requested, carrying no stop (it reads through into the native TGA).

- **Re-cut protection (PAM-blocking).** g3 needs none (the insertion splits its target). For g2, a single **silent** change in the 5' arm (codon 389 GGG->GGT, both Glycine) destroys the NGG PAM; the CDS translation is unchanged and g2 can no longer cut the donor or edited allele. One donor therefore serves either guide.

- **Restriction note.** The insert (linker+mCherry) is BsaI/BsmBI-free. The genomic homology arms contain 2 BsaI and 1 BsmBI site(s) (native TP53 sequence); these do not affect the requested re-clonability of mCherry but should be noted if Golden Gate assembly of the whole donor is planned.

## 6. Genotyping and validation strategy

Three primer pairs are provided; all genomic primers are unique on hg38 (isPcr) and the logic was confirmed by in-silico PCR on WT, donor and knock-in templates:

- **5' junction PCR** (1171 bp): forward outside the 5' arm (genomic) + reverse in the insert. Product only on correct 5' integration -- no product on WT or on the donor plasmid.

- **3' junction PCR** (1370 bp): forward in the insert + reverse outside the 3' arm (genomic). Confirms 3' integration; KI-specific.

- **Internal / zygosity pair** (across the insert): **WT 262 bp vs knock-in 1015 bp**, distinguishing wild-type / heterozygous / homozygous clones and providing a Sanger amplicon across the whole insert and both junctions.

Anchoring the junction primers outside the homology arms is deliberate: the donor carries only ~800 bp of arm, so a primer beyond the arm cannot prime on residual donor -- a band proves genuine genomic integration.

Note: the internal/zygosity pair sits **within** the homology arms, so it also amplifies residual donor plasmid/dsDNA (same knock-in-size band). Treat the **junction PCRs as the definitive integration proof** and account for donor carryover when scoring zygosity.

## 7. Critical caveats and recommendations

**(a) HEK293T expresses SV40 large T antigen -- a confound for p53 dynamics.** 293T cells stably express SV40 large T antigen, which binds p53 directly, **stabilizes** it (raising basal levels) while **inhibiting its transactivation** and blocking p53-dependent MDM2 induction (Lilyestrom 2006). p53 stress dynamics (stabilization pulses, target-gene feedback) in 293T are therefore not representative of normal p53 signaling. If the goal is physiological p53 dynamics, a p53-wild-type line without a viral oncoprotein (e.g. MCF7, RPE1-hTERT, A549, U2OS, HCT116) is strongly preferable. If HEK293T is required, interpret dynamics with the large-T caveat in mind. **This is the single most important point in this report.**

**(b) C-terminal tag vs p53 regulation.** The CTD (363-393) governs MDM2-dependent turnover and PTM-based regulation. Validate that tagged p53 still: stabilizes after genotoxic stress (e.g. doxorubicin, nutlin-3a), localizes to the nucleus, and induces canonical targets (CDKN1A/p21, MDM2) -- ideally against an untagged control.

**(c) HEK293 ploidy.** HEK293/293T is hypotriploid (~3 copies of chr17/TP53). Expect multiple alleles to edit; the internal/zygosity PCR and Sanger reads will reveal allele dosage. Homozygous (all-allele) tagging may require screening more clones.

**(d) Delivery & HDR.** Deliver the donor as a plasmid or long dsDNA (arms are 800 bp -- too long for ssODN). Co-deliver SpCas9 + g3 sgRNA (RNP or plasmid). HDR is enhanced by cell-cycle synchronization and small-molecule NHEJ inhibition if needed. Validate with the junction PCRs, then Sanger across the insert, then confirm mCherry fluorescence and tagged-p53 function.

**(e) Multiplex/specificity scope.** Specificity here is for the human genome (host). No multiplex panel applies. The guide cloning oligos provided are for pX330/pSpCas9(BB)-family BbsI vectors.

## 8. Methods, databases and auditability

TP53 NM_000546.6 retrieved from NCBI; genomic sequence from the local GRCh38/hg38 2bit. Stop codon anchored by exact sequence match (build-independent). Guides enumerated with a custom script; on-target and specificity scores from CRISPOR v5.2 (hg38; batch retained); exhaustive off-targets from Cas-OFFinder v2.4.1 (hg38, CPU) and, independently, FlashFry against a purpose-built hg38 SpCas9-NGG database (cached locally for reuse). mCherry codon optimization with DNAChisel (h_sapiens table) enforcing BsaI/BsmBI absence. Primers with primer3; specificity by isPcr against hg38; binding-site variant screen via Ensembl Variation (GRCh38). Data retrieved 2026-06-29/30. The numbered scripts/ pipeline ships as the reproducible record.

## 9. Output files

- `TP53_mCherry_Knockin_Report.pdf` -- this report

- `TP53_mCherry_Knockin_Design.xlsx` -- guides, off-targets, donor components, primers, cloning oligos

- `sequences.fasta` -- guides, mCherry/insert, donor + arms, primers, BbsI cloning oligos

- `figures/` -- knock-in strategy; specificity evidence; donor + genotyping

- `scripts/` -- reproducible numbered pipeline (ships with the project)

## 10. References

1. O'Leary NA, et al. (2016). Reference sequence (RefSeq) database at NCBI. *Nucleic Acids Res* 44:D733-D745. TP53 transcript NM_000546.6.
2. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from *Discosoma* sp. red fluorescent protein. *Nat Biotechnol* 22:1567-1572. (mCherry; GenBank AY678264)
3. Concordet JP, Haeussler M (2018). CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. *Nucleic Acids Res* 46:W242-W245.
4. Bae S, Park J, Kim JS (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. *Bioinformatics* 30:1473-1475.
5. Doench JG, et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. *Nat Biotechnol* 34:184-191. (CFD / Rule Set 2)
6. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013). Genome engineering using the CRISPR-Cas9 system. *Nat Protoc* 8:2281-2308.
7. Lilyestrom W, Klein MG, Zhang R, Joachimiak A, Chen XS (2006). Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor. *Genes Dev* 20:2373-2382.
8. Kruse JP, Gu W (2009). Modes of p53 regulation. *Cell* 137:609-622. (C-terminal domain: ubiquitination, acetylation, PTMs)
9. Lahav G, Rosenfeld N, Sigal A, Geva-Zatorsky N, Levine AJ, Elowitz MB, Alon U (2004). Dynamics of the p53-Mdm2 feedback loop in individual cells. *Nat Genet* 36:147-150.
