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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2485:1–13. doi: 10.1007/978-1-0716-2261-2_1

Table 1.

A summary of CRISPR screening technologies applicable to pooled CRISPR sgRNA library screening.

Function Type of Cas9 Targeting
Location		 Notes
Knock-Out spCas9 First exon Cas9 derived from S. pyogenes is DNA endonuclease used to induce frame-shift mutations at the targeted locus, resulting in gene inactivation
Activation dCas9 fused to VP64 Promoter VP64 is a tetrameric repeat of the herpes simplex virus protein VP16, which induces transcriptional gene activation [23, 24]
Activation dCas9 fused to p300 Promoter p300 is a histone acetyltransferase which facilitates gene transcription [25]
Activation dCas9 fused to PRDM9 Promoter PRDM9 is a histone methyltransferase used to stabilize gene expression via induction of the activating mark H3K4me3
Inhibition or activation dCas9 fused to HDAC3 Promoter HDAC3 is a histone deacetylase associated with both gene activation and repression depending on the targeted locus [26]
Inhibition dCas9 fused to Dnmt3a Promoter or CpG islands Dnmt3a is a DNA methyltransferase used to induce targeted DNA methylation and suppress gene transcription [27, 28]
Inhibition dCas9 fused to KRAB Promoter or enhancer Kruppel-associated box domain (KRAB) recruits a complex responsible for both histone methylation and deacetylation, resulting in heterochromatin formation and repression of gene transcription [29-31]
Inhibition dCas9 fused to KRAB–MeCP2 Promoter or CpG islands KRAB in combination with methyl CpG binding protein 2 (MeCP2) aids in gene silencing via complex formation with histone deacetylases and via direct interaction with transcription factors [32]
Inhibition dCas9 fused to LSD1 Promoter LSD1 is a histone demethylase used to repress enhancers by removing H3K4me2 mark from histone, resulting in reduced gene expression due to enhancer inactivation [33]
Mutagenesis dCas9 fused to AIDx Gene or regulatory region AID is activation-induced cytidine deaminase with the ability to generate a wide array of targeted point mutations in high-throughput screens for disease-related variants [34, 35]