Discussion
The identification of host genes contributing to viral entry is an important step in understanding the pathogenesis of viruses and identify in new therapeutic strategies. Several whole genome CRISPR screens against live SARS-CoV-2 identified host factors contributing to infection (Baggen et al., 2021; Daniloski et al., 2021; Schneider et al., 2021; Wang et al., 2021; Wei et al., 2021). Here we have devised a strategy using lentiviral pseudoviruses focusing on the selection strategy on the viral entry step at a level compatible with biosafety level-2 laboratories.
For the analysis of SARS-CoV-2 entry, the lentiviral pseudovirus is advantageous as it has a similar size and shape ~100 nm in diameter (Illanes-Álvarez et al., 2021; Rein, 2019), making it compatible with similar endocytic mechanisms. While, VSV based pseudovirus has also been used for SARS-CoV-2 screens (Wei et al., 2021), this strategy has the limitation that VSV is larger than SARS-CoV-2 and has a bullet shape that is ~200 nm in length (Cureton et al., 2010). Our study, however, did not determine if this difference results in any changes in the viral entry.
In addition to improving the stringency for CRISPR screens on viral entry, the use of the CRE-Gag fusion protein focuses the gene hits onto entry related processes rather than genes affecting reporter expression. This is because CRE reporter can be activated directly by the CRE recombinase protein provided by the viral particles without the need of the integration process (Esposito et al., 2016; Philippe E. Mangeot et al., 2019).