Dense sgRNA Library Construction Using a Molecular Chipper Approach

Jijun Cheng; Wen Pan; Jun Lu

doi:10.21769/BioProtoc.2373

Dense sgRNA Library Construction Using a Molecular Chipper Approach

Bio Protoc. 2017 Jun 20;7(12):e2373. doi: 10.21769/BioProtoc.2373.

Authors

Jijun Cheng^{1

2}, Wen Pan^{1

2}, Jun Lu^{1

2

3

4}

Affiliations

¹ Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.
² Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA.
³ Yale Cooperative Center of Excellence in Hematology, Yale University, New Haven, Connecticut, USA.
⁴ Yale Center for RNA Science and Medicine, Yale University, New Haven, Connecticut, USA.

Abstract

Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA.

Keywords: CRISPR-Cas9; Molecular chipper; Non-coding genome; Reporter screen; sgRNA library.

Abstract

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