CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled CRISPR screens

Abstract

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are incompatible with pooled screening methodologies that require mRNA-embedded barcodes, including some microscopy and single cell sequencing approaches. Here, we report the development of CROPseq-multi, a CROPseq-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with mRNA-embedded barcodes. CROPseq-multi has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. CROPseq-multi is compatible with enrichment screening methodologies and optical pooled screens, and is extensible to screens with single-cell sequencing readouts. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode detection efficiency 10-fold, enables detection of recombination events, and increases decoding efficiency 3-fold relative to CROPseq. CROPseq-multi is a widely applicable multiplexing solution for diverse SpCas9-based genetic screening approaches.

Genetic interaction library screening with a next-generation dual guide CRISPR system

“…Interestingly, when we analysed cloning of a larger library of over 100,000 guides (see below, Figure 1G), we found the rate of undesired guide pairs was much lower, at only 1.95% in the cloned library, with 88.8% reads mapping to the designed guide pairs. This improvement is likely to be due to developments in the oligo pool synthesis and cloning methodology including reduction in template concentration during the initial PCR to 20 pg/μl, as has recently been observed elsewhere [26]. The lower recombination rates could also result from a different composition of the library, which is an anchor-library design (Methods) meaning that there are fewer replicates of the same guide and that recombination events would more frequently recreate other guide pairs in the library.…”

“…Our methodology can also be applied to delivery of guide pairs for other applications such as genetic deletion libraries [32] to identify functional non-coding regions of the genome, or to deliver pairs of prime editing and nicking guide RNAs into the same cell for prime editing screens of defined mutations [33]. Dual guide approaches will also make detection of guide RNAs in single cell [34,35] or pooled optical CRISPR screens [26] more sensitive, which is frequently limiting in primary or stem-cell derived cells. Our system is also amenable to improving single gene knockout by targeting the same gene with two guides to make a SuperMinLib [5,15] or improving CRISPR activation or inhibition efficiency [36,37].…”