read_status: read
type: paper
title: Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
year: 2014
authors:
- Shalem, Ophir
- Sanjana, Neville E.
- Hartenian, Ella
- Shi, Xi
- Scott, David A.
- Mikkelsen, Tarjei S.
- Heckl, Dirk
- Ebert, Benjamin L.
- Root, David E.
- Doench, John G.
- Zhang, Feng
journal: Science
doi: 10.1126/science.1247005
url: https://www.science.org/doi/10.1126/science.1247005
project:
- IGP
system:
- human_cells
- cancer_cell_lines
- genome
disease:
- melanoma
- cancer_drug_resistance
genes:
- cas9
- nf1
- nf2
processes:
- genome_wide_loss_of_function
- dna_double_strand_break_repair
- positive_selection_screen
- negative_selection_screen
methods:
- pooled_crispr_screen
- lentiviral_sgRNA_delivery
- deep_sequencing_quantification
- gene_set_enrichment_analysis
key_findings:
- high_efficiency_gene_knockout
- identification_of_essential_genes
- identification_of_drug_resistance_genes
- improved_specificity_over_rnai
limitations:
- incomplete_frameshift_penetrance
- off_target_editing_risk
- cell_line_specificity
relevance:
- enabling_functional_genomics
- scalable_loss_of_function_screening
- therapeutic_target_discovery
concepts:
- genome_scale_crispr_screening
- positive_vs_negative_selection_logic
- crispr_vs_rnai_perturbation
tags:
- crispr
- functional_genomics
- screening_methods
Improving Whole-Genome Screens Improved methods are needed for the knockout of individual genes in genome-scale functional screens.Wang et al.(p.80, published online 12 December) and Shalem et al.(p.84, published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods., Genome-editing technology allows improved positive or negative selection screens.
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
CRISPR–Cas9 enables efficient genome-scale loss-of-function screening in human cells
DNA-level knockout yields stronger phenotypes than partial knockdown approaches
Negative selection screens recover core essential gene programs
Positive selection identifies genes whose loss confers drug resistance
High concordance across independent sgRNAs supports causal inference
Lentiviral delivery of Cas9 + sgRNA in a single vector
Pooled sgRNA libraries targeting constitutive exons
Low-MOI transduction to ensure single perturbations per cell
Deep sequencing of sgRNA abundance for phenotype inference
GSEA and RIGER-based hit ranking
Establishes CRISPR screening as a replacement for RNAi in mammalian cells
Reframes functional genomics around complete gene inactivation
Demonstrates scalability to diverse phenotypes beyond viability
Introduces selection logic transferable to many disease contexts
Figure 1: Validation of efficient CRISPR-mediated gene knockout versus RNAi
Figure 2: Genome-scale negative selection identifies essential genes
Figure 3: Positive selection screen identifies vemurafenib resistance genes
Figure 4: Direct comparison of CRISPR and shRNA screening performance
Not all indels generate null alleles
Off-target cleavage not fully eliminated
Screens limited to proliferative phenotypes
Findings dependent on cellular context
Systematic dissection of noncoding regulatory elements