Forward Genetic Screens of Human Glycosylation Pathways Using the GlycoGene CRISPR Library. Issue 4 (15th April 2022)

• Record Type: Journal Article
• Authors: Kelkar, Anju; Groth, Theodore; Neelamegham, Sriram
• Is Part Of: Current protocols. Volume 2:Issue 4(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-04-15
• Subjects: CRISPR‐Cas9 -- glycogene -- glycosylation -- lectin -- pathway analysis; Life sciences -- Laboratory manuals -- Periodicals; Biology -- Laboratory manuals -- Periodicals; Life sciences -- Technique -- Periodicals; Biology -- Technique -- Periodicals; 570.028
• DOI: 10.1002/cpz1.402
• Languages: English
• ISSNs: 2691-1299

Abstract

Abstract: CRISPR‐Cas9‐based forward genetic screens represent a powerful discovery platform to uncover genes regulating specific biological processes. This article describes a method for utilizing a freely available GlycoGene CRISPR library to knock out any gene participating in human glycosylation in arbitrary cell types. The end product is a stable GlycoGene CRISPR knockout cell library, where each cell contains one or more sgRNA and lacks corresponding function. The cell library can be screened using various lectin/antibody reagents. It can also be applied in functional assays to establish glycan structure‐glycogene‐glycopathway relationships. This is a powerful systems glycobiology strategy for dissecting glycosylation pathways and processes. © 2022 Wiley Periodicals LLC. Basic Protocol 1 : Scale‐up and NGS validation of the GlycoGene CRISPR plasmid library Basic Protocol 2 : Preparation of a GlycoGene CRISPR lentivirus pool and an isogenic cell line stably expressing Cas9 nuclease Basic Protocol 3 : Preparation of a GlycoGene CRISPR cell library, self‐inactivation of Cas9, and library validation by NGS Basic Protocol 4 : Enrichment of lectin‐binding or non‐binding cells and related multiplex NGS data acquisition Basic Protocol 5 : Bioinformatics pathway analysis