Abstract
BackgroundTargeted 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.ResultsWe 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.ConclusionsClonal 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.
Highlights
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
Stable genetic alteration can be achieved by integration of overexpression and short hairpin RNA (shRNA) constructs [2], or by genomic editing of the endogenous protein locus with transcription activator-like effector nucleases (TALENs) [3], zinc-finger nucleases [4, 5], and, more recently, by RNA-guided nucleases based on the clustered, regularly interspaced
We describe a simple method for isolation of clonal deletion mutants based on CRISPR/Cas9 targeting combined with dot immunoblot analysis and validated by western blots and DNA sequencing
Summary
Targeted genomic editing using the CRISPR/Cas methodology has opened exciting new avenues in probing gene function in virtually any model system, including cultured mammalian cells. Transient perturbations involve chemical inhibition (and activation) with small molecules, overexpression from nonintegrating vectors, or knockdown by RNA interference [1]. Stable genetic alteration can be achieved by integration of overexpression and shRNA constructs [2], or by genomic editing of the endogenous protein locus with transcription activator-like effector nucleases (TALENs) [3], zinc-finger nucleases [4, 5], and, more recently, by RNA-guided nucleases based on the clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas system [6,7,8,9,10,11].
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