CRISPR/Cas9 technology enables rapid generation of loss-of-function mutations in target genes in mammalian cells. Single cells carrying these mutations can be used to establish new cell lines to create CRISPR-induced knockout clones. These clonal cell lines are important tools for exploring protein function, analyzing the consequences of gene loss, and studying the specificity of biological agents. However, successful derivation of knockout clones can be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clone heterogeneity.
The efficiency of each CRISPR system is limited by how efficiently it can be introduced and expressed in target cells. In the two-vector approach, lentiviruses are first used to stably transduce a Cas9-expressing plasmid into a target cell line. After selecting for Cas9 expression, this line can easily be used to generate multiple knockout clones by expressing different guide RNAs. However, constitutive Cas9 expression may result in higher levels of off-target mutagenesis, and some cell lines tend to silence virally expressed proteins.
In the single-vector approach, a single Cas9/gRNA plasmid can be introduced into cells by transfection or transduction. However, the size of proviruses generated from all-in-one vectors approaches lentiviral packaging limits, which may reduce subsequent viral titers. In general, if researchers are trying to analyze different gene knockouts in the same cell line, it is recommended to use a two-vector approach and generate stable Cas9-expressing cell lines. If researchers are looking to analyze individual gene knockouts and minimize off-target effects, transient transfection with poly-in-one vectors may be better. Many laboratories have developed CRISPR plasmids with convenient drug resistance and fluorescent labeling.
CRISPR can be used to interfere with mammalian gene function in a variety of ways. CRISPR-mediated homology-directed repair can be used to replace wild-type alleles with mutations of interest, as described elsewhere. Alternatively, if the goal of the experiment is simply to eliminate gene function, it can be achieved by targeting Cas9 to this locus and relying on the cell’s NHEJ pathway to repair DSBs with indel mutations. A population of cells can be transfected or transduced with a CRISPR construct at a high multiplicity of infection (MOI), resulting in mutations in most cells. However, this approach may yield heterogeneous cell populations: some cells may escape target modifications entirely and can tolerate certain mutations without compromising protein function.
These CRISPR knockout cell lines are suitable for a variety of downstream applications and can be used to study fundamental questions in cell biology, genetics, and cancer biology. For example, in a recent study, CRISPR was used to generate isogenic human cell line models in which different DNA repair genes were deleted. The lines were allowed to grow in culture for up to 1 month, and the “mutation signature” caused by each knockout was determined by whole-genome sequencing. These hypermutation patterns were then compared to mutational signatures found in human tumors, allowing the researchers to experimentally verify the genetic basis of these motifs.
In another recent study, CRISPR was used to identify transporters that import the amino acid serine into mitochondria. The researchers first performed a CRISPR screen to identify guide RNAs that caused poor growth, particularly in serine-deficient media. The researchers then generated cell lines knocking out SFXN1, a hit of the screen, and found that these lines lacked mitochondrial serine input. These examples illustrate how CRISPR-induced gene knockout allows precise interrogation of the relationship between genotype and phenotype, thereby revealing the genetic architecture of mammalian cells.
CRISPR-generated knockout cell lines can also be used to study the specificity of drugs, antibodies, and other biological reagents. Nonetheless, there are multiple potential defects that interfere with the generation and analysis of CRISPR knockout cell lines. Some CRISPR-induced mutations can lead to incomplete target ablation through exon skipping and/or nonsense-associated alternative splicing (NAS). In these little-known processes, the presence of nonsense mutations causes cells to produce alternative transcripts that bypass CRISPR-induced mutations. Although the transcripts produced may differ from the predominant isoform expressed in the cell line, they may still be sufficient for the function of the protein.
https://www.creative-biogene.com/crispr-cas9/
