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Related Experiment Video

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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

Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.

Emmanouil Metzakopian¹, Alex Strong¹, Vivek Iyer¹

¹Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Scientific Reports

|May 24, 2017

Summary

This summary is machine-generated.

This study introduces novel CRISPR-Cas9 genome-wide sgRNA libraries for human and mouse research. These tools enable precise gene editing and functional screening, accelerating biological discoveries.

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Published on: April 8, 2017

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

CRISPR-Cas9 technology has become a standard tool in biological research, surpassing conventional gene editing methods.
Its applications span both genome-wide and gene-specific investigations, highlighting its versatility.

Purpose of the Study:

To present the first individually cloned CRISPR-Cas9 genome-wide arrayed sgRNA libraries for human and mouse.
To provide a comprehensive and flexible gene editing toolbox for diverse research applications.

Main Methods:

Development of arrayed sgRNA libraries for 17,166 human and 20,430 mouse genes.
Cloning of sgRNAs into a lentivirus backbone with PiggyBac transposase elements, fluorescent, and drug selection markers.
Validation of sgRNA efficacy using CEL-1 assays for DNA cleavage and FLAER labeling for loss-of-function mutations.

Main Results:

Libraries cover 34,332 human and 40,860 mouse sgRNAs.
Over 95% of tested sgRNAs demonstrated specific DNA cleavage.
sgRNAs targeting GPI anchor protein pathway genes successfully induced loss-of-function mutations in cell lines.

Conclusions:

The developed arrayed libraries facilitate high-throughput screening of individual genes, gene combinations, and large gene sets.
These libraries enable rapid deconvolution of results from genome-wide screens.
This comprehensive gene editing toolbox offers significant scientific value for biological research.