In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9

• Published in: Developmental biology 2023-12, Vol.504, p.120-127
• Main Authors: Tiyaboonchai, Amita, Wakefield, Leslie, Vonada, Anne, May, Catherine L., Dorrell, Craig, Enicks, David, Sairavi, Anusha, Kaestner, Klaus H., Grompe, Markus
• Format: Article
• Language: English
• Subjects: Animals; CRISPR-Cas Systems - genetics; Integrases - genetics; Keratin-14 - genetics; Keratin-14 - metabolism; Mice; Mice, Transgenic; Promoter Regions, Genetic - genetics; RNA Polymerase II - genetics; RNA Polymerase II - metabolism
• ISSN: 0012-1606; 1095-564X; 1095-564X
• DOI: 10.1016/j.ydbio.2023.09.011

The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on RNA polymerase II promoter driven self-cleaving guide RNAs (scgRNA) to achieve tissue-specificity. To demonstrate proof-of-principle for this approach a transgenic mouse was generated harbouring a knock-in of a scgRNA into the Cytokeratin 14 (Krt14) locus. Krt14 expression marks the stem cells of squamous epithelium in the skin and oral mucosa. The scgRNA targets a Stop cassette preceding a fluorescent reporter in the Ai9-tdtomato mouse. Ai9-tdtomato reporter mice harbouring this allele along with a spCas9 transgene demonstrated precise marking of the Krt14 lineage. We conclude that RNA polymerase II promoter driven scgRNAs enable the use of CRISPR/spCas9 for genetic lineage tracing. [Display omitted] •Cell-type specific expression of CRISPR self-cleaving guide RNAs (scgRNAs) enables in vivo genetic lineage tracing.•Reporter activation by scgRNA knock-in to the Cytokeratin 14 locus recapitulates results from Cre-based lineage tracing.•Inducible expression of the Cytokeratin 14 reporter was achieved with doxycycline inducible spCas9.