Introduction
The CRISPR-Cas9 system, which can be programmed to target specific genomic loci (position), offers a powerful tool for gene editing and functional genomics. This study demonstrates developing and applying a genome-scale CRISPR-Cas9 knockout (GeCKO) library for high-throughput screening (the use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level) to identify genes essential for various cellular functions and disease mechanisms.
Methods
- GeCKO Library Construction: The GeCKO library was designed to target 18,080 genes in the human genome using 64,751 unique guide RNA (gRNA) sequences. This library was delivered into human cells via lentiviral vectors that also included Cas9 and a puromycin resistance marker to select successfully converted cells.
- Cell Lines and Screening: Two cell lines were used for screening:
- The A375 melanoma cell line is derived from malignant tumors, the cell line produces rapidly growing melanomas.
- The HUES62 human embryonic stem cell line is derived from human embryos. It is a versatile tool for biomedical research. I go more in-depth in the video.
Results
- Validation of GeCKO Library:
- EGFP Knockout Experiment: Using a cell line expressing enhanced green fluorescent protein (EGFP), the authors demonstrated efficient gene knockout, with a high percentage of cells losing fluorescence after transduction with EGFP-targeting gRNAs.
- Negative Selection Screening: The library was used to identify essential genes for cell viability. Deep sequencing after 14 days revealed a significant depletion of gRNAs targeting essential genes, confirmed through gene set enrichment analysis; a method to identify classes of genes or proteins that are over-represented in a large set of genes or proteins.
Discussion
- Comparison with RNAi: The authors highlight several advantages of CRISPR-Cas9 over RNA interference (RNAi):
- CRISPR-Cas9 creates permanent gene knockouts by introducing indels in the DNA, while RNAi reduces gene expression at the mRNA level, often leading to incomplete knockdown.
- CRISPR-Cas9 can target non-coding regions, allowing for the study of regulatory elements in addition to coding genes.
- Off-Target Effects: While off-target effects were observed, the authors suggest that these can be minimized using techniques like paired nickases or high-fidelity Cas9 variants.
Conclusion
The study establishes CRISPR-Cas9 as a robust tool for genome-scale functional screening, capable of identifying both essential genes and those involved in specific cellular responses, such as drug resistance. The high efficiency and consistency of the CRISPR-Cas9 system make it a promising approach for large-scale genetic studies and therapeutic target discovery.
Resource:
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014 Jan 3;343(6166):84-87. doi: 10.1126/science.1247005. Epub 2013 Dec 12. PMID: 24336571; PMCID: PMC4089965.
6/25/2024
