Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection

Abstract

Background: Targeted genomic editing using the CRISPR/Cas9 methodology has opened exciting new avenues in probing gene function in virtually any model system, including cultured mammalian cells. Depending on the desired mutation, several experimental options exist in the isolation of clonal lines, such as selection with introduced markers, or screening by PCR amplification of genomic DNA. However, streamlined approaches to establishing deletion and tagging mutants with minimal genomic perturbation are of interest in applying this methodology. Results:We developed a procedure for rapid screening of clonal cell lines for the deletion of a protein of interest following CRISPR/Cas9 targeting in the absence of selective pressure based on dot immunoblots. To assess the technique, we probed clonal isolates of 293-TREx cells that were targeted with three separate sgRNAs against the HuR gene. Validation of knockout candidates by western blot indicated that the normalized protein abundances indicated by the dot blot serve as accurate predictors of deletion. In total, 32 independent biallelic deletion lines out of 248 screened clones were isolated, and recovery of null mutants ranged from 6 to 36 % for the individual sgRNAs. Genomic sequencing verified small deletions at the targeted locus. Conclusions:Clonal screening for CRISPR/Cas9-mediated editing events using dot immunoblot is a straightforward and efficient approach that facilitates rapid generation of genomic mutants to study gene function.

Frizzled 6 disruption suppresses osteoblast differentiation induced by nanotopography through the canonical Wnt signaling pathway

“…Given that the ultimate goal was the generation of an FZD6 knockout, the protein expression analysis ensured that the method was successful, avoiding the unnecessary study of deletions or in-frame mutations (Estep, Sternburg, Sanchez, & Karginov, 2016 (Boyan et al, 2018).…”

Application of CRISPR/Cas9 in Understanding Avian Viruses and Developing Poultry Vaccines

“…In this experiment cells are infected with the recombinant viral vector harboring the antigen. Characterization is achieved through the implementation of 3 major steps: (1) protein separation from the mixture through SDS-PAGE, (2) transfer of separated protein into a solid support such as nitrocellulose membrane, and (3) detection of the protein through binding to appropriate antibodies (Estep et al, 2016). To further characterize the expression of the antigen protein, immunofluorescence or cell imaging is employed.…”

HuR controls apoptosis and activation response without effects on cytokine 3’ UTRs

“…To evaluate HuR's effect on cytokine expression through their 3′ UTRs, full-length UTR sequences were cloned into a dual-luciferase reporter vector. Initially, a panel of six cytokine UTRs was screened in the easily transfectable human embryonic kidney T-REx-293 cells and a derived HuR KO cell line [48]. Previously, we have demonstrated a significant, albeit mild, regulation of an AUUUAcontaining reporter by HuR using these lines [49].…”

“…Knockouts in Jurkat cells were obtained by selection-free immunoblot screening as previously described [48]. Jurkat cells were cultured in RPMI1640 media with 10% FBS and 1x penicillin/streptomycin at 37°C with 5% CO 2 .…”