CRISPR/Cas9-mediated Genomic Editing of Cluap1/IFT38 Reveals a New Role in Actin Arrangement

Abstract

CRISPR/Cas9-mediated gene editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interactions and in contrast to exogenous expression of a protein, this can be studied maintaining physiological stoichiometry, topology, and context. We have used CRISPR/Cas9-mediated genomic editing to investigate Cluap1/IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis, which was then correlated to the corresponding interactome determined by ectopic expression. Besides IFT-B complex components, new interacting proteins like Ephrin-B1 and TRIP6, which are known to be involved in cytoskeletal arrangement and protein transport, were identified. With the identification of platelet-derived growth factor A (PDGFA) and coiled-coil domain-containing protein 6 (CCDC6) two new interactions were discovered, which link Cluap1 to ciliogenesis and cancer development. The CRISPR/Cas9-mediated knockout of Cluap1 revealed a new phenotype affecting the actin cytoskeleton. Together, these data provide first evidence for a role of Cluap1 not only for cilia assembly and maintenance but also for cytoskeletal rearrangement and intracellular transport processes.

Keywords: Affinity proteomics; CRISPR/Cas9; Cellular organelles*; Cluap1; Knockouts*; Protein complex analysis; Protein-Protein Interactions*; cilia assembly; gene editing; intraflagellar transport.

Fig. 1.

Experimental workflow. For overexpression of Strep/FLAG-tagged Cluap1 isoforms and the empty Strep/FLAG vector as control, SILAC-labeled Hek293T cells were transfected. After 48 h, cells were lysed and Strep affinity purification was performed. The combined eluates (differentially labeled control and isoforms of one experiment, respectively) were trypsin digested and analyzed by mass spectrometry. CRISPR/Cas9-generated endogenously FLAG-tagged Hek293T cells were grown in normal DMEM/10%FBS/2.5% penicillin/streptomycin medium. FLAG affinity purification was followed by trypsin digestion and mass spectrometry. For both, overexpression experiments and endogenously tagged cells, MaxQuant data were statistically analyzed using Perseus (see also “Experimental Design and Statistical Rationale,” 31). In parallel, Cluap1 hTERT-RPE1 knockout cells were generated by CRISPR/Cas9 targeting exon 1 and exon 6 for double strand break. Single clones were investigated by fluorescence staining for cilia and cytoskeletal markers and for behavior e.g. migration rate.

Fig. 2.

Protein complex analysis using endogenously tagged Cluap1. (A) The mass spectrometry data of five biological replicates were statistically analyzed using MaxQuant and Perseus (highlighted: proteins with significance A < 0.05 and student's t test p < 0.05). The whole IFT-B complex could be detected in both, C- and N-terminally tagged Cluap1 HEK293T cells (blue). (B) Mass spectrometry data were validated by Western blotting. Using a Cluap1 antibody, two isoforms were detected in N- but only one in C-terminally tagged cells. (C) The SILAC-labeling approach was used to investigate protein complex formation of single Cluap1 isoforms. With overexpression of the isoform 1 some IFT-B proteins, the BBSome component BBS7 and new factors, e.g. CCDC6 and CEP55, were enriched (17 biological replicates, highlighted: proteins with significance A < 0.05 and student's t test p < 0.05).

Fig. 3.

No cilia and an increase of actin filaments in Cluap1 Ex1 and Ex6 KO. The cell phenotype was observed either under complete (+FBS, A–C) or starving condition (-FBS, D–H). Cilia were stained with ARL13B (red), and actin was visualized in green using Phalloidin-Alexa488 (A–H). Cilia were absent in Cluap1Ex1 KO and Ex6 KO hTERT-RPE-1 cells (E, F) whereas control cells showed normal cilia assembly after 24 h of starvation (D). Phalloidin-Alexa488 staining revealed a high amount of filamentous actin in KO (B, C, E, F) compared with control cells (A, D). Ectopic expression of the IFT-B complex binding Cluap1 isoform 1 could rescue the ciliary assembly but not the actin organization (G–I) (I) Nuclei and cilia of two independent experiments at 5–7 positions per sample were counted, and the percentage of ciliated cells was calculated. The total number of counted cells is shown at the bottom of each bar. No cilia were detected in both KO cell lines.

Fig. 4.

Migration was slowed down in Cluap1Ex1 and Ex6 KO cells. (A–C) Control and KO sells were incubated in starvation medium for 24 h prior to scratch. Images of two independent experiments with three biological replicates at three positions each were captured after scratch (t0, A–C) and 7 h later (t7, A′–C′). (D) The cell-free area was measured using ImageJ Fiji and the percentage of closure was calculated. The significance was calculated using the Student's t test. (E) The proliferation rate was measured using the crystal violet assay. There was no significant difference in cell number at any time point between control and KO cells.