lentiGuide-Puro

Genome-wide, targeted loss-of-function hybrid screens using CRISPR-Cas9 in human and mouse cells provide an alternative screening system for RNA interference (RNAi). Previously, researchers used genome-scale CRISPR knockout (GeCKO) libraries to identify loss-of-function mutations that confer resistance to vemurafenib in melanoma models. However, lentiviral delivery systems initially used for CRISPR screening either had low viral titers or required cell lines that already expressed Cas9, thus limiting the range of biological systems suitable for screening. Researchers sought to improve lentiviral packaging and guide sequence selection in the original GeCKO library. To create a new vector capable of producing higher titer virus (lentiCRISPRv2), they made several modifications, including removal of one of the nuclear localization signals, human-codon optimization of the remaining nuclear localization signal and P2A bicistronic linker sequences, and repositioning of the U6-driven sgRNA cassette (Figure 1a). These changes resulted in an approximately tenfold increase in functional viral titers compared to lentiCRISPRv1 (Figure 1b).

To further increase viral titers, the researchers also cloned a dual-vector system in which Cas9 (lentiCas9-Blast) and sgRNA (lentiGuide-Puro) were delivered using separate viral vectors with different antibiotic selection markers (Figure 1a). The functional viral titer of lentiGuide-Puro increased approximately 100-fold over the original lentiCRISPRv1 (Figure 1b). Both single- and dual-vector systems mediate efficient knockout of genome-integrated EGFP copies in human cells. While the two-vector system is capable of generating Cas9-expressing cell lines that can subsequently be used for screening using lentiGuide-Puro, the single-vector lentiCRISPRv2 may be more suitable for in vivo or primary cell screening applications.

Figure 1. CRISPR lentiviral vectors with higher functional titer. (Sanjana, Neville E., et al., 2014)