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

Dev Biol. 2023 Oct 7:S0012-1606(23)00165-3. doi: 10.1016/j.ydbio.2023.09.011. Online ahead of print.ABSTRACTThe 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.PMID:37813160 | DOI:10.1016/j.ydbio.2023.09.011

Source: Developmental Biology - October 9, 2023 Category: Biology Authors: Amita Tiyaboonchai Leslie Wakefield Anne Vonada Catherine L May Craig Dorrell David Enicks Anusha Sairavi Klaus H Kaestner Markus Grompe Source Type: research