Table 1.

Summary table of advantages and disadvantages for each of the three screening methods discussed; CRISPR, RNAi and overexpression screening

	Advantages	Disadvantages
CRISPR/Cas9	Permanent targeting of genes for robust response. Ability to multiplex. Ease of construction. Adaptability of 20 bp protospacer. Target specificity. Eliminates confounding effects from low level protein expression. Use of dCas9. Reduces activation of innate immune response compared with RNAi. Well established in mammalian cell culture.	Cannot be used to study essential genes. Relies on Cas9 expression levels. Requires selection step. Difficult to identify moderately acting antiviral factors. Off-target effects. Redundancy.
RNAi	Ability to investigate essential genes. Ability to restore protein expression for validation. Reagents readily available. Well established in mammalian cell culture, mice, and Drosophila models.	Off-target effects and high rate of false-positives. Low percentage of reproducible hits. Inability to knockdown non-coding regions. Incompleteness of gene knockdown. Longer siRNAs can trigger the immune response. Redundancy.
Overexpression	Combined species libraries (human and macaque). Suitability for arrayed comparative screening. Library limited to ISGs. Able to identify moderately acting antiviral factors.	Dependent on producing high lentiviral stocks. ISG-mediated and overexpression artefact toxicity. Requirement for fluorescence readout. Requirement for automation of equipment. Cannot identify proteins that function in a complex. ‘Long genes’ more difficult to handle.