![]() As with other genetic mutant approaches, it is beneficial to isolate several independently derived knockout clones to control for secondary mutations. We wished to apply the CRISPR/Cas system to establish stable cell lines completely lacking specific protein function. The approach can also be applied to screening for insertions of tags mediated by homologous recombination. Screening directly for protein production ensures proper knockout, avoiding signal from in-frame deletions/mutations that may not eliminate its function. In the present study, 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. In a common approach, purification of genomic DNA from isolated clones permits PCR amplification followed by sequencing, deletion analysis or restriction digest. However, the relatively high efficiency of Cas9 targeting allows for clonal screening without selection. Furthermore, it leaves a significantly-sized, transcribed insertion that may affect the expression of nearby genes, although it may be excised through an additional Cre-mediated step, if surrounded by loxP sites. While this approach is beneficial if the sgRNA has low efficiency, or if the mutation confers a strong selective disadvantage to the cells, it requires the construction of repair template vectors containing the marker and homology arms specific to the locus. In the targeted modification strategies, the researcher has the option to isolate correct clones by introducing a selectable marker at the genomic locus. sgRNA/Cas9 targeting to a gene exon induces a double-stranded DNA break that is repaired by non-homologous end joining, often introducing frameshift mutations (depicted in red) that lead to lack of functional protein production and likely cause nonsense-mediated decay of the mRNA Aside from NHEJ-mediated deletions, the Cas cleavage can also stimulate homology-driven repair based on a supplied DNA template, and allow for larger deletions or insertion of tags and markers. In contrast, small in-frame deletions may not compromise the function of the protein. Deletions that target the open reading frame and result in frame-shifts, particularly early in the mRNA, are very likely to yield a non-functional protein sequence and to target the mRNA for nonsense-mediated decay due to premature stop codons in the new frame (Fig. A small, random deletion is often introduced at the repair site. In its simplest form, creation of a knockout line depends on the sgRNA-guided dsDNA cleavage by the Cas endonuclease, followed by non-homologous end joining (NHEJ) repair of the site by the cell. Thus, targeting of nearly any genomic sequence is possible with the introduction of an sgRNA and Cas9 into the cells of interest. Due to its particularly simple makeup, the type II CRISPR/Cas system of Streptococcus pyogenes has been adapted for genomic editing with great success: a single protein, Cas9, is required for crRNA binding and cleavage, and the RNA components have been engineered into a single guide RNA (sgRNA). Its transcription and processing yields small crRNAs that associate with and guide CRISPR-associated (cas) protein(s) to complementary DNA targets for endonucleolytic cleavage. Short (20–30 bp) sequence tags from the invaders are incorporated as spacers between direct repeats of a CRISPR locus. The CRISPR/Cas module is an endogenous adaptive immunity system commonly used by bacteria and archaea to counteract phage infection and introduction of plasmid DNA. Combinations of stable integration and modification with temporal control include the use of chemically inducible systems, such as tetracycline repression/activation, tamoxifen control of Cre-ER recombination, and more modular control of expression, localization and activity by small molecules. Often more experimentally desirable, stable genetic alteration can be achieved by integration of overexpression and shRNA constructs, or by genomic editing of the endogenous protein locus with transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and, more recently, by RNA-guided nucleases based on the clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Transient perturbations involve chemical inhibition (and activation) with small molecules, overexpression from non-integrating vectors, or knockdown by RNA interference. Manipulating protein levels and activities is a principal tool in understanding the functions and the relationships of these molecular components in cells.
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