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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

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
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Compact CRISPR genetic screens enabled by improved guide RNA library cloning.

Seok-Jin Heo1,2, Lauren D Enriquez1,2, Scot Federman1,2

  • 1Laboratory for Genomics Research, San Francisco, CA, 94158, USA.

Genome Biology
|January 19, 2024
PubMed
Summary

This study introduces a new CRISPR library cloning protocol that enhances sgRNA uniformity and reduces bias. This method enables efficient genome-wide CRISPR screens in challenging cell models with fewer cells.

Keywords:
CRISPR screeningGuide libraryLibrary cloningPrimary cell CRISPR screeningiPSC-derived CRISPR screening

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Last Updated: Jun 15, 2026

Pooled CRISPR-Based Genetic Screens in Mammalian Cells
00:09

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Published on: September 4, 2019

21.9K
CRISPR Guide RNA Cloning for Mammalian Systems
06:48

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Published on: October 2, 2018

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A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
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A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

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Area of Science:

  • * Genomics
  • * Molecular Biology
  • * Genetic Engineering

Background:

  • * CRISPR genome editing offers potential for defining human gene function in specific cell types.
  • * Existing genome-wide libraries face challenges in efficient genetic perturbation in relevant models.
  • * Need for improved methods to overcome limitations in CRISPR screening.

Purpose of the Study:

  • * To develop an improved library cloning protocol for CRISPR genome-wide screens.
  • * To increase single-guide RNA (sgRNA) uniformity and reduce bias in existing libraries.
  • * To enable efficient CRISPR screens in technically challenging cell models.

Main Methods:

  • * Development of a novel library cloning protocol for CRISPR sgRNA construction.
  • * Assessment of sgRNA uniformity and bias reduction in the new libraries.
  • * Comparison of statistical power and cell requirements with previous screening methods.

Main Results:

  • * The new protocol significantly increases sgRNA uniformity and reduces library bias.
  • * CRISPR screens using the improved libraries achieved equivalent or superior statistical power.
  • * The method requires an order of magnitude fewer cells compared to previous screens.
  • * Enabled genome-scale CRISPR screens in challenging cell models.

Conclusions:

  • * The developed cloning protocol enhances the efficiency and reduces bias in CRISPR genome-wide libraries.
  • * This advancement facilitates robust CRISPR screening in difficult-to-engineer cell types.
  • * The improved protocol supports broader applications of CRISPR screens in biological research.