A design was requested for an endogenous C-terminal mCherry knock-in at TP53 (NM_000546.6, GRCh38) in HEK293T cells using SpCas9 and an HDR donor — guide RNAs at the stop codon cross-validated across multiple tools, an HDR donor with ~800 bp homology arms, a GS linker and PAM-blocking silent mutations, junction genotyping primers, and a full off-target report, with the mCherry human codon-optimized and free of internal BsaI/BsmBI sites. The delivered package provides a primary and backup guide, a 2,353 bp HDR donor (800/800 bp arms, (GGGGS)x3 linker, codon-optimized 708-nt mCherry), 5'/3' junction and internal/zygosity genotyping primers, and a three-independent-tool off-target report, alongside a PDF report, an Excel workbook, annotated sequences (FASTA), and figures. The primary guide cuts just 6 bp from the insertion point and is intrinsically re-cut-immune — its protospacer is split by the inserted cassette, so no PAM-blocking mutation is needed — and across Cas-OFFinder, CRISPOR and FlashFry it shows zero off-targets at <=1 mismatch and no hit in any TP53 pseudogene or p53-family gene, directly clearing the off-target failure mode that affected the previous design; no common SNP (MAF >= 1%) lies under either guide's binding site. The single most important caveat is biological rather than technical: HEK293T constitutively expresses SV40 large T antigen, which binds and functionally inactivates p53, so p53 stress dynamics in this line are non-physiological — a p53-wild-type line without a viral oncoprotein (e.g. MCF7, RPE1-hTERT, A549, U2OS, HCT116) is preferable if the goal is physiological p53 dynamics, and tagged-p53 function should be validated experimentally.
