IntroductionUnlike Cas9, Cas12a possesses intrinsic RNase activity, enabling CRISPR-RNA (crRNA)-array maturation from single transcripts, simplifying multiplexed orthogonal gene editing and regulation1,2. Cre-dependent CRISPR systems provide control over CRISPR-protein3,4,5,6 or guide RNA (gRNA) expression5,7. Controlling only the gRNA enables sequential gene targeting7,8 in addition to acting as a simple on/off switch. However, dependency on external sources of Cre-recombinase exposes such switches to limitations of the Cre/LoxP-system, including cytotoxicity from constitutively expressed wild-type Cre9, while inducible variants like CreERT210 suffer from leakiness11,12 and inefficiency13,14. A CRISPR-inducible Cre-system has the potential to be a transformative addition to the CRISPR toolbox, offering a recombinase-based solution that, as of now, has remained elusive. Here we present the development of a CRISPR inducible recombinase we term SWITCHER from its initial validation to a stepwise evolution into a self-integrating CRISPR switch facilitating sequential execution of distinct CRISPR programs.ResultsDesign and the proof-of-concept of SWITCHER variantsWe explored the idea of utilizing a floxed wild-type Cre construct as a compact and mobile genetic switch under the control of the CRISPR/Cas12a system capable of modulating Cas12a activity in return.However, the caveat of this construct lies in its dynamic nature, preventing it from existing within a cellular context. To overcome this obstacle, SWITCHER is assembled from two non-functional split-Cre fragments15 separated by a CRISPR target site with a 162 bp overlap serving as homology arms (HA) for precise DNA repair. Activation occurs via a DNA-Strand-Break (DSB) between the Cre-fragments, likewise a previously described single-strand annealing (SSA) dependent reporter assay16. In the case of SWITCHER, SSA-mediated correction of the open reading frame results in Cre expression and, subsequently, removal of its own origin. Non-targeted or targeted SWITCHER constructs repaired by alternative mechanisms (e.g. non-homologous end-joining (NHEJ)) are trans-converted within the same cell (Fig. 1a, Fig. S1).Fig. 1: SWITCHER, a Cas12a-inducible wild-type Cre acting as a digital switch.a Scheme of the SWITCHER construct (1), mechanism of activation (2), and recombination acting as a digital switch (3). b DsRed expression three days after co-transfection of HEK293T cells with SWITCHER-TF1-DsRed and either dCas12a or Cas12a and specific (crF1) or non-targeting (crR2) crRNAs (3 plasmids required for activation). The positive control is CreERT2 (2 plasmids required). Scale bars, 50 μm. c Flow cytometry-based DsRed quantification of cells from (b) showing the percentage, fold change, and median fluorescence intensity. Data are shown as mean ± s.d. using data from n = 3 biological replicates. Ordinary one-way ANOVA followed by Šídák’s multiple comparisons test. Calculated P-values were depicted as follows: ****p