Table 2.

Features of the different perturbation tools used for targeted genetic screens

	Loss of function			Gain of function
	Cas9 nuclease	CRISPRi	RNAi tools	CRISPRa	cDNA overexpression
Type of perturbation	Indel mutation in the target DNA that generally results in a complete knockout owing to a coding frameshift	Repression of gene expression by dCas9-mediated transcriptional inhibition	Repression of gene expression by targeting the mRNA molecule for degradation and translational inhibition	Activation of gene expression by dCas9-mediated recruitment of transcriptional activation domains to TSSs	Exogenous overexpression of cloned cDNA constructs
Expected off-target effects	Additional unexpected indels in the genome	Repression of additional genes and effects on chromatin	Repression of additional mRNAs owing to partial ‘seed’ matching and imprecise Dicer processing; global effects owing to saturation of endogenous RNAi machinery (mostly relevant to siRNA transfections)	Expression of additional genes and effects on chromatin	Not many gene-specific off-target effects; global effects on translation owing to strong expression of a single gene
On-target efficacy	With continuous expression, near-complete allelic modification can be achieved in a short time frame	Inhibition level depends on the choice of sgRNA and the basal expression level of the target gene	Repression efficacy depends on the choice of RNAi tool and the specific targeting sequence	Activation level depends on the choice of sgRNA and the basal expression level of the target gene	High expression of most cDNA constructs owing to expression from the same promoter
Constitutive versus conditional expression	Cas9 expression can be made conditional	Cas9 expression can be made conditional	Only Pol II-driven RNAi reagents can be conditionally expressed	Cas9 expression can be made conditional	cDNA constructs can be conditionally expressed
Reversibility of perturbation	Irreversible	Reversible	Reversible	Reversible	Reversible
Refs	44–47	48	1	48,49	69

dCas9, catalytically inactive Cas9; Pol II, RNA polymerase II; RNAi, RNA interference; sgRNA, single guide RNA; siRNA, small interfering RNA; TSS, transcription start site.