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CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

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Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Published on: September 4, 2019

Genome-scale CRISPR-Cas9 knockout screening in human cells.

Ophir Shalem^1,2, Neville E Sanjana^1,2, Ella Hartenian¹

¹Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.

Science (New York, N.Y.)

|December 17, 2013

Summary

This study introduces a genome-scale CRISPR-Cas9 knockout (GeCKO) library for efficient gene function screening. The technology successfully identified essential genes and drug resistance factors in human cells, showcasing its potential for biological discovery.

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Published on: April 8, 2017

Area of Science:

Genomics
Molecular Biology
Gene Editing

Background:

CRISPR-Cas9 nuclease allows precise genomic modification.
Genome-scale screening offers a novel approach to study gene function.

Purpose of the Study:

To develop and validate a genome-scale CRISPR-Cas9 knockout (GeCKO) library for high-throughput screening.
To identify genes critical for cell viability and drug resistance in human cells.

Main Methods:

Lentiviral delivery of a GeCKO library targeting 18,080 genes.
Negative and positive selection screening in human cancer and pluripotent stem cells.
Screening for genes involved in vemurafenib resistance in a melanoma model.

Main Results:

Identified genes essential for cell viability in cancer and stem cells.
Discovered novel genes (NF2, CUL3, TADA2B, TADA1) and validated known genes (NF1, MED12) associated with vemurafenib resistance.
Demonstrated high consistency and hit confirmation rates for the GeCKO library.

Conclusions:

The GeCKO library enables efficient genome-scale functional genomics screening.
CRISPR-Cas9 screening is a powerful tool for identifying genes involved in cellular processes and therapeutic resistance.