Screen Follow-Up
A powerful approach to follow up on an initial pooled screen is to create a secondary pooled library that targets many fewer genes than the original library but does so with more perturbations per gene. For example, if the primary screen was conducted with a genome-wide library with 80,000 guides (4 per gene), a secondary library may target 500 genes with 8 guides per gene. This results in a 20-fold reduction in library size, with a total of 4,000 guides.The increase in guides per gene provides greater statistical confidence in identifying true positives, rescuing false negatives, and discarding false positives, while the smaller overall size of the library facilitates screens across more conditions (e.g. additional cellular models, more drug doses, or readouts of increased-complexity).
Such a custom library is easily designed with CRISPick: Designing a Pooled Library with CRISPick.
Another approach to consider is a combinatorial library to understand the genetic interaction network of hit genes. Here, Cas12a is particularly useful, due to its ease of multiplexing. This is supported by CRISPick, and we have additional advice on combinatorial libraries: Designing a Multi-Guide Cas12a Library.
Top hit genes of particular interest can be dissected at near-amino-acid-level resolution via the use of base editor tiling libraries: Designing a Base Editor Library with Beagle.
Additionally, we support synthetic ORF libraries that introduce all amino acid changes at all possible positions: Screening with Deep Mutational Scanning.
Individual reagents are available as bacterial streaks, DNA, and/or small-scale virus preps:
- Ordering sgRNAs
- human-target guides for CRISPR knockout
- Ordering shRNAs
- human and mouse shRNAs for RNAi-based knockdown
- Ordering ORFs
- human ORFs for overexpression
Custom cloning of individual (arrayed) reagents in 96-well plate format is also available.