Pooled lentiviral libraries with a barcoded sgRNA expression cassette that is expressed and can be detected using RNA Sequencing
Pooled CRISPR screening with lentiviral sgRNA libraries has proven to be a very effective approach to identify genes functionally required to generate particular phenotypes, and RNA-Seq is an effective method to find the underlying changes in gene expression producing those phenotypes. With the advent of droplet microfluidic platforms that enable single-cell molecular analysis on a large scale, these two technologies may be merged to assess and characterize the distinct expression profiles produced by genetic disruption in a pooled CRISPR knockout screen. With this sort of pooled screening approach using CRISPR barcode libraries, isolation and characterization of phenotype-specific cells is unnecessary.
CloneTracker XP CRISPR Barcode Libraries express an sgRNA for pooled CRISPR genetic screens with a unique molecular identifier (UMI or barcode) that facilitates clonal cell tracking and single-cell expression analysis. In these libraries, the barcoded sgRNA expression cassette is positioned so that it is on the 3′ non-coding region of the puromycin selection gene and present on the polyA+ when this gene is expressed. As a result, the sgRNA and barcode can be detected in the RNA expression data. When combined with single-cell expression profiling, changes in gene expression can be directly correlated to gene knockout, knockdown, or activation, depending on the type of CRISPR construct used to make the library.
Cell-specific barcodes incorporated into pooled CRISPR sgRNA libraries may be used in conjunction with single-cell RNA expression analysis to perform what is sometimes called a Perturb-Seq or CRISPR-Seq screen. The approach enables researchers to reliably identify and link changes in gene expression to the knockout of particular target genes.