Project Planning

The overall goal of the GPP is to accelerate the field of functional genomics, the toolkit of experimental approaches for understanding gene function. To fulfill this mission, we partner with scientists in the Broad community and beyond on their biological question of interest, engaging in the project from conception to analysis to publication.

Our expertise includes inherent challenges, pitfalls, and optimization guidelines that are not always obvious - this technical information, and years of experience seeing common challenges across projects, often makes the difference between a good idea and a successful experiment. Continued engagement will also ensure that a project has access to cutting-edge tools and methodologies we develop far in advance of publication.

Reviewing our GPP Collaboration Guide and scheduling an introductory meeting with one or more GPP screening scientists is a good place to start. Below is a depiction of a typical collaboration using a pooled screening approach, but please note that we also support single-cell RNA sequencing and optical pooled screening readouts.

Reagents

To aid in conceptualizing a screen, we provide an overview of our most commonly-used reagents. Click on ‘Inventoried’ or ‘Customized’ to read more about GPP's reagents. Please note that this is not an exhaustive list and further discussion with your screening scientist(s) is often helpful.

Inventoried

Customized

GPP maintains a collection of pooled CRISPR libraries across modalities - knockout, activation, and interference - as well as a human open reading frame (ORF) library:

• Selecting a Genome-Wide Library: Guidance on genome-wide libraries and considerations across the numerous CRISPR modalities
• Druggable CRISPR Libraries: Additional details for a suite of libraries targeting genes annotated as druggable from the Pharos project

A full list of existing pooled libraries: Browse existing libraries

Instructions for acquiring these reagents can be found here: Ordering Pooled Libraries

GPP also maintains individual (arrayed) reagents that can be provided as bacterial streaks and/or plasmid DNA and/or lentivirus:

• sgRNAs: Largely for CRISPR knockout
• shRNAs: Our collection of RNAi reagents
• ORFs: Human Open Reading Frames

GPP can also generate customized libraries, and then clone those libraries into a wide variety of existing vectors, which can be browsed using dimgarF, or a new vector can be designed using Fragmid:

Designing a Pooled Library with CRISPick

Supports multiple Cas enzymes and modalities.

Designing a Multi-Guide Library

Designing a Multi-Guide Cas12a Library - For multiple perturbations per cell, whether targeting the same gene or multiple genes, using Cas12a.

Designing a Dual-Guide Cas9 Library - For two perturbations per cell, whether targeting the same gene or multiple genes, using an SpCas9-SpCas9 or CROPseq-Multi-v2 system.

For studying genes of interest at near-amino-acid-level resolution via base editor-directed endogenous mutagenesis.

Screening with Deep Mutational Scanning

Deep Mutational Scanning (DMS) technology enables high resolution interrogation of a protein of interest by probing every possible amino acid substitution at every position. In a DMS screen, a library of cDNAs encoding the protein variants is synthesized, incorporated into an expression cassette, and ectopically expressed.

Additional information on ordering a customized library is found here: Order custom sgRNA pooled libraries.

Custom cloning of individual (arrayed) reagents in 96-well plate format is also available: Ordering 96-well Custom Constructs

FAQs:

Q: If thinking about an in vivo screen, are there additional resources?

Indeed. Maintaining coverage in an in vivo setting often requires specific experimentation. Cell Counter Libraries can be used for this purpose.

Alternatively, you may consider adding iBar UMIs to your library.

Project PlanningAssay DevelopmentScreeningPost-Screen AnalysisScreen Follow-UpSummary Tables