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).