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Homologous Recombination

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

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Updated: Mar 5, 2026

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

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

Published on: December 6, 2017

Flexible CRISPR library construction using parallel oligonucleotide retrieval.

Abigail Read¹, Shaojian Gao², Eric Batchelor³

¹Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.

Nucleic Acids Research

|March 24, 2017

Summary

This summary is machine-generated.

We developed optimized CRISPR/Cas9 gene knockout libraries for human and mouse functional screens. Our method enables cost-effective, customized library generation for enhanced functional genomics applications.

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Substrate Generation for Endonucleases of CRISPR/Cas Systems

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Published on: September 8, 2012

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Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

Area of Science:

* Molecular Biology
* Genomics
* Genetic Engineering

Background:

* CRISPR/Cas9 gene knockout libraries are essential for functional genomic screens.
* Existing libraries may have variable on-target potency and off-target effects.
* Customization of libraries is often desired but can be resource-intensive.

Purpose of the Study:

* To present pre-designed human and mouse single guide RNA (sgRNA) sequences optimized for high on-target activity and low off-target effects.
* To describe a cost-effective method for constructing customized CRISPR libraries.
* To facilitate functional genomics research by providing accessible resources.

Main Methods:

* Curation of sgRNA sequences targeting constitutive 5' exons and conserved protein domains for maximal gene inactivation.
* Design of a robust method for constructing small-sized CRISPR libraries from array-synthesized oligonucleotide pools.
* Utilization of parallel oligonucleotide retrieval for library construction.

Main Results:

* A set of pre-designed sgRNA sequences for human and mouse CRISPR/Cas9 libraries with high on-target potency and low off-target effects.
* A scalable and cost-effective method for generating customized CRISPR libraries.
* Demonstrated feasibility of creating variable-sized libraries with controlled coverage depth.

Conclusions:

* The developed sgRNA resources and construction method offer a convenient solution for individual laboratories.
* These tools empower researchers to generate tailored CRISPR libraries for specific functional genomics applications.
* This work enhances the accessibility and efficiency of CRISPR-based functional screening.