CRISPR/Cas9-mediated target validation of the splicing inhibitor Pladienolide B

Abstract

CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

Keywords: AB, Alamar Blue; CRIPSR/Cas9; CRISPR, Clustered Regulatory Interspaced Short Palindromic Repeats; Cas9, CRISPR associated protein; DSB, double strand break; Drug discovery; Drug target validation; HR, Homologous Recombination; NHJE, Non-Homologous End-Joining; PB, Pladienolide B; Pladienolide B; Pre-mRNA splicing; SSNs, Site Specific Nucleases; Spliceosome; T7EI, T7 Endonuclease I; TALENs, Transcription Like Effector Nucleases; ZFN, Zing Finger Nucleases; sgRNA, Guide RNA.

Fig. 1

Detection of the targeted modification of each spliceosome subunit by T7EI assay and Sanger sequencing. (A–D) T7EI assays and alignment of Sanger sequencing reads of PCR amplicons encompassing the targets of SF3b1, SF3b2, SF3b3 and SF3a3 genes respectively. SgRNAs targets are highlighted in red, the PAM sequence is underlined, dashes indicate nucleotide deletions, nucleotides highlighted in blue indicate insertions, and nucleotides highlighted in green indicate substitutions. Arrow indicates the expected size of the DNA bands of corresponding amplicons cleaved by T7EI. (E) Genome editing efficiency in each subunit estimated by the number of mutant clones divided by the total number of sequenced clones.

Fig. 2

Effect of the specific gene editing on the toxicity of the antitumor macrolide Pladienolide B on the HEK293T cells. (A) Cell viability was assessed by colorimetric assay using Alamar Blue Cell Viability Reagent (Life Technologies, UK). HEK293T cells edited in various genes (SF3A3, SF3B1, SF3B2, and SF3B3) and treated with 100 nM of Pladienolide B for 24 h at 37 °C humidified incubator with 5% CO₂. Specific deletion of SF3B1 displayed high level of resistance to the PB when compared to the un-transfected cells or cells transfected only with wtCas9, or sgRNA or dCas9. Result is a representative of three independent experiments and the bars represents the mean ± SD and *P < 0.01.