Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Abstract

CRISPR/Cas9 technology enables the rapid generation of loss‐of‐function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR‐induced knockout clone. 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. 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. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. 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. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.

A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles

“…These relatively small deletions (<300 bp) generate minimal genome perturbation when compared with approaches using a pair of gRNAs targeting sequences located in different exons many kilo-bases apart, resulting in deletion of large intragenic regions, in less predictable editing outcome and in possible interference with gene regulatory sequences. [32][33][34] This strategy is amenable to the study of essential genes, as the cells can be used for biological assay or to derive omics datasets quite early after the CRISPR process (i.e., prior to the induction of cell death). It is also applicable to primary cells, especially when using synthetic gRNAs that are less cytotoxic since they do not induce a strong antiviral response.…”

Base Editing in Human Cells to Produce Single‐Nucleotide‐Variant Clonal Cell Lines

“…This can be accomplished by using more than two plates per transfection. This protocol uses an adaptation of methods previously described (Giuliano, Lin, Girish, & Sheltzer, 2019). The use of the strainer is optional, but when using it, we have observed a higher frequency of isolating single-cell-derived colonies as opposed to doublet-cell-derived colonies.…”

Pooled CRISPR screening at high sensitivity with an empirically designed sgRNA library

“…Overall, comparing genes identified to be essential using either BAGEL or MAGECK RRE revealed strong agreement with few essential genes private to each software (Supplementary In a final step, we asked how differences in editing efficiency, the number of sgRNAs per gene as well as potential clonality effects might affect the determination of gene essentiality. We were especially interested in differences in gene essentiality between the Cas9 bulk cell line and the two Cas9 single cell clones populations, since the generation of single cell clones from bulk populations forces cells to go through a genetic bottleneck, which might favor certain genetic alterations (Giuliano et al 2019) and thus dependencies. Therefore, we used BAGEL (Hart and Moffat 2016) to analyze depleted genes in each HAP1 cell line using the combined HD CRISPR library (8 sgRNAs per gene) and the individual sub-libraries A and B.…”