(A) AlphaFold2 model of the π-EB1 tetramer (Mirdita et al., 2022). Note that AlphaFold2 does not correctly predict relative domain positions and did not capture the LOV2/Zdk1 interaction correctly although a structure of the LOV2/Zdk1 dimer has previously been determined (Wang et al., 2016). (B) Overview of the one-step CRISPR/Cas9-mediated insertion of a π-element construct containing the photosensitive LOV2/Zdk1 module, a fluorescent protein marker, and an internal EF1α promoter. Arrows indicate the location of PCR primers. Lowercase letters indicate mutations introduced to make the homology-directed repair (HDR) template resistant to Cas9 cleavage. (C) Genomic PCR to validate π-element integration into the endogenous EB1 locus with primers as indicated in (B). The two clones shown are homozygous as there is no short product in PCR2, which corresponds to the non-edited EB1 locus. (D) Immunoblots with antibodies as indicated of control and π-EB1 i3N clones before and after 2 days of neuron differentiation showed replacement of EB1 by the photosensitive π-EB1 variant and expected +TIP expression level changes associated with neuron differentiation. (E) RT-qPCR analysis of the expression levels of the π-element N- and C-terminal halves relative to EB1 expression in wild-type (Ctrl) i3N hiPSCs. Shown are the mean and data points from individual qPCR reactions. (F) Comparison of nuclear Oct4 staining (white) as a pluripotency marker in control and π-EB1 i3N hiPSC colonies. Nuclei are identified with DAPI (blue). (G) Image of a π-EB1 i3N hiPSCs colony with magnified images on the right showing dissociation of EGFP-Zdk1-EB1C from growing MT ends in blue light. (H) Immunoblot of control and π-EB1 and EB3-/- i3Neurons showing expression of π-EB1 and deletion of both EB1 and EB3. (I) π-EB1 i3N hiPSCs transiently expressing a mScarlet-tagged EB1N MT-binding domain before and during blue light exposure. Maximum intensity projections in alternating green and magenta over 20 s at 3 s intervals illustrate attenuation of MT growth during blue light exposure. (J) Quantification of the median MT growth rate per cell before and during blue light exposure in control and π-EB1 i3N hiPSCs. Gray lines connect data points from the same cell. Statistical analysis by paired t-test for each i3N hiPSC line.