Summary-Paper 10

23 Jan 2023
by Richa
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Summary- paper 10: A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Alina Guna, Katharine R. Page, Joseph R. Replogle, Theodore K. Esantsi, Maxine L. Wang, Jonathan S. Weissman, Rebecca M. Voorhees

Biorxiv, 2023

Questions/gaps addressed:

  • Establishing a pipeline that permits querying epistatic relationships with a variety of phenotypic readouts in any cells expressing the CRISPRi machinery.

Key methods:

  • CRISPR interference (CRISPRi) based approach with a flexible dual-sgRNA CRISPRi-v2 library (Addgene #83969; 5 sgRNA/gene library targeting human protein-coding genes) compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens.

  • Construct and deliver a library containing a fixed pre-determined guide, genetic ‘anchor point’, and a second randomized CRISPRi guide from a single lentiviral backbone at scale.

  • Two parallel screens, one with a ‘non-targeted contol’ and the other target gene as ‘ achor point’.

  • FACS-based approach, adapted a fluorescent split GFP reporter system to look at relationships between the parallel pathways specifically to query insertion into the ER. Constitutively expressed the first 10 β-strands of GFP (GFP1-10) in the ER lumen and appended the 11th β -strand onto the C-terminal of the endogenous sequence of SEC61β (SEC61β-GFP11). Engineered K562 cells to stably express ER GFP1-10 and the dCas9-KRAB(Kox1) CRISPRi machinery. Inducible promoter, integrated the SEC61β-GFP11 reporter alongside a normalization marker (RFP) separated by a viral 2A sequence, allows to use the GFP:RFP ratio to differentiate transcriptional and translational differences.

Major takeaways:

  • Approach eliminates the need to create and characterize a knock-out line for a particular gene of interest, to set up epistatsic genetic interation screens.

  • Construction of new libraries is easy/fast, with a two-step cloning process,uses existing sequencing and analysis pipelines.

  • Caveat: addition of a second guide delivered on the same plasmid diminishes the efficiency of the fixed guide. Alternative using Zim3-Cas9 effector system, with stronger on-target knockdown compared to KOX1-Cas9 while maintaining minimal non-specific genome-wide effects.