title: 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.
authors.affiliation: Department of Cell Biology and Neuroscience, Institute for Integrative Genome Biology, University of California, Riverside, CA, 92521, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Estep
authors.affiliation: Department of Cell Biology and Neuroscience, Institute for Integrative Genome Biology, University of California, Riverside, CA, 92521, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Sternburg
authors.affiliation: Department of Cell Biology and Neuroscience, Institute for Integrative Genome Biology, University of California, Riverside, CA, 92521, USA.
authors.forename: Gissell A
authors.initials: GA
authors.lastname: Sanchez
authors.affiliation: Department of Cell Biology and Neuroscience, Institute for Integrative Genome Biology, University of California, Riverside, CA, 92521, USA. karginov@ucr.edu.
authors.forename: Fedor V
authors.initials: FV
authors.lastname: Karginov
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D000067780:ELAV-Like Protein 1 / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods; D005838:Genotype; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D000067780:ELAV-Like Protein 1; D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas9; Clonal selection; Dot blot; Screening