Fig. 1. Durable gene knockdown by CRISPR interference in human iPSC-derived neurons.

(A) Construct pC13N-dCas9-BFP-KRAB for the expression of CRISPRi machinery from the CLYBL safe-harbor locus: catalytically dead Cas9 (dCas9) fused to blue fluorescent protein (BFP) and the KRAB domain, under the control of the constitutive CAG promoter.

(B) Timeline for sgRNA transduction, selection and recovery, doxycycline-induced neuronal differentiation and functional analysis of CRISPRi-i³N iPSCs.

(C) Knockdown of the transferrin receptor (TFRC) in CRISPRi-i³N iPSCs and neurons. CRISPRi-i³N iPSCs were lentivirally infected with an sgRNA targeting TFRC or a non-targeting negative control sgRNA. Neuronal differentiation was induced by addition of doxycycline on Day -3 of the differentiation protocol and plating cells in neuronal medium on Day 0. Cells were harvested at different days for qPCR. After normalizing by GAPDH mRNA levels, ratios of TFRC mRNA were calculated for cells expressing the TFRC-targeting sgRNA versus the non-targeting sgRNA; mean ± SD (two biological replicates).

(D, E) Knockdown of ubiquilin 2 (UBOLN2) in CRISPRi-i³N neurons. CRISPRi-i³N neurons infected with UBQLN2 sgRNA or non-targeting control sgRNA were harvested on Day 11 for qPCR (D) or Western blot (E) to quantify UBQLN2 knockdown at the mRNA level or protein level, respectively. (D) Relative UBQLN2 mRNA level was determined by normalizing UBQLN2 mRNA level by GAPDH. Relative UBOLN2 mRNA was calculated for cells expressing the UBOLN2-targeting sgRNA versus the non-targeting sgRNA; mean ± SD (three biological replicates). (E) Left, representative Western blot (Loading control β-Actin). Right, quantification of UBQLN2 protein levels normalized by β-Actin for cells with non-targeting sgRNAs or UBOLN2 sgRNA; mean ± SD (two independent Western blots).

(F,G) Knockdown of progranulin (GRN) in CRISPRi-i³N neurons. CRISPRi-i³N neurons infected with GRN sgRNA or non-targeting control sgRNA were harvested on Day 11 for qPCR (F) or monitored by immunofluorescence (IF) microscopy on Day 5. (G) Relative GRN mRNA level normalized by GAPDH mRNA. Ratio of relative GRN mRNA for cells expressing the GRN-targeting sgRNA versus the non-targeting sgRNA; mean ± SD (three biological replicates). (G)Top row, non-targeting negative control sgRNA. Bottom row, sgRNA targeting progranulin. Progranulin signal (IF, green), neuronal marker Tuj1 (IF, red) and nuclear counterstain DRAQ5 (blue) are shown.