TABLE 1

Comparison of CRISPRa to other activation methodsa

Activation methodActivated geneEase of productionThroughput of productionUsed in pooled genome-wide screensOff-target effectsBlocked by methylated DNAExpression of mutant alleles or specific splice variantsLimitations in useNo. of components requiredReferences
CRISPRaEndogenousEasy (simple sgRNA cloning)HighYesMinimalNoCan target specific variants only if different variants have different TSSs; not ideal for expressing mutant allelesRequires an “NGG” PAM adjacent to a target sequence2 or 3 (dCas9, sgRNA, optional activation module); these may be collected on one vector in some cases3, 4, 24, 26, 29, 30, 32, 44
ORF overexpressionExogenously addedMedium to difficult (entire ORF of gene must be cloned)LowYes, but libraries are burdensome to create and can contain biases toward smaller ORFs or certain splice variantsNonexistentNAYesLonger or GC-rich genes can be difficult to clone145, 4649
TALE or ZFEndogenousMedium to difficult (requires complicated cloning [TALEs] or protein engineering [ZFNs])LowNoMinimalTALEs can be blocked by methylated DNA but can also recognize it specificallyCan target specific variants only if different variants have different TSSs; not ideal for expressing mutant allelesTALEs have no sequence limitations. ZFNs may require some engineering to target a given sequence139, 50, 51, 5253, 5456
  • a NA, not applicable; TSS, transcription start site.