To knock out a gene of interest, the nuclease Cas9 and a target-specifying guide RNA must be delivered to the cell. STRATEGIC PLANNING Choosing a Knockout Strategy
CLONE X CROSS REFERENCE VERIFICATION
The procedure includes (1) choosing a knockout strategy (2) selecting gRNA target sites and performing vector cloning (Support Protocols 1– 3, Basic Protocol 1) (3) introducing CRISPR plasmids by transfection or transduction (Basic Protocols 2– 5) (4) isolation and expansion of single-cell clones (Basic Protocol 6, Alternate Protocol 1) and (5) knockout verification by western blot analysis, PCR, and/or Sanger sequencing (Support Protocol 4, Basic Protocols 7– 9). Schematic outline of the knockout process. Knockout verification by western blot analysis, PCR, and/or Sanger sequencing. Isolation and expansion of single-cell clones
Introducing gRNAs by transfection or transduction
Selecting gRNA target sites and performing vector cloning The protocol is divided into five sections, as outlined in Figure 1: Below, we describe an efficient method to use CRISPR to generate knockout clones in mammalian somatic cell lines. In this way, CRISPR can be used to introduce stable, nonrevertible alterations to mammalian genes. Alternately, the cell can repair the lesion via nonhomologous end joining (NHEJ), an error-prone process that commonly results in an insertion or deletion (indel) mutation at the DSB location (Brinkman et al., 2018). If a suitable template is provided, the cell can use homology-directed repair to integrate a novel allele or transgene at the targeted site (Ceasar, Rajan, Prykhozhij, Berman, & Ignacimuthu, 2016). The cell then has multiple options for repairing that break. By expressing the Cas9 nuclease and a suitable guide RNA (gRNA) in mammalian cells, a double-strand break can be introduced at a locus of interest. CRISPR has been particularly useful in the study of mammalian genetics and cell biology, as mammalian somatic cells have historically proven to be highly refractory to genetic modification (Komor, Badran, & Liu, 2017). It has since been co-opted by scientists as a means to generate sequence-specific double-strand breaks (DSBs) and to induce other precise alterations in the genomes of cells and organisms (Cong et al., 2013). The CRISPR system initially evolved as a nucleic acid–targeting bacterial defense mechanism capable of conferring resistance to viral infection (Barrangou et al., 2007). These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells.
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