Fig. 4: ProDomino confidently predicts potent opto- and chemogenetic Cas9 and Cas12a variants.

a,d, Insertion scores predicted by ProDomino are mapped onto the primary sequences of Cas9 (a) or Cas12a (d). Selected high- and low scoring sites are marked in purple and gray, respectively. b,e, The insertion scores predicted by ProDomino are mapped onto experimentally resolved structures of Cas9 (b) and Cas12a (e). Insertion sites selected for experimental validation are indicated (PDB ID 4UN3 and 6IV6). f, Zoomed-in views of the insertion sites of the two Cas12a lead candidates. c, HEK293T cells were transfected with vectors encoding (1) the indicated Cas9/VPR-LOV hybrid variant (or Cas9/VPR as control), (2) a TetO targeting sgRNA together and a Renilla luciferase and (3) a firefly luciferase preceded by multiple TetO repeats. Samples were incubated under blue light or in the dark for 48 h, followed by luciferase assay. g–i, HEK293T cells were transfected with vectors encoding (1) the indicated Cas12a–GR2 hybrid variant (or wild-type Cas12a as control), and (2) a gRNA targeting the endogenous RUNX (g), GRIN2B (h) or VEGFA (i) locus. Samples were treated with cortisol or DMSO as indicated. 72 h posttransfection, InDel frequencies were assessed by next-generation sequencing. c,g–i, Bars indicate means, error bars the standard deviation and black dots individual data points from n = 2 (h) or n = 3 (c,g,i) independent experiments. Cor, cortisol.