Fig. 2: Overview of CRISPR-based genome and epigenome engineering tools.
From: CRISPR-based functional genomics tools in vertebrate models
a Gene knockout or replacement: Cas9 nuclease creates DSBs at target sites guided by sgRNA. Repair occurs via NHEJ, leading to small insertions or deletions that disrupt gene function, or via HDR using a donor DNA template to enable precise gene correction or insertion. b ABE: a catalytically impaired Cas9 (nCas9) fused to an adenosine deaminase converts A•T base pairs to G•C through A-to-I (inosine) editing, which is read as G during DNA replication or repair. c CBE: nCas9 is fused to a cytidine deaminase and often paired with UGI. This enables C-to-T editing, converting C•G base pairs to T•A. d Transcriptional repression (CRISPRi): a catalytically dead Cas9 (dCas9) is fused to a KRAB repressor domain and guided to promoter regions, where it inhibits gene transcription without cutting DNA. e Transcriptional activation (CRISPRa): dCas9 is fused to a transcriptional activator and directed to a gene’s promoter or enhancer to initiate transcription. f Epigenome editing: dCas9 is tethered to epigenetic modifiers (for example, methyltransferases) to install or erase epigenetic marks such as DNA methylation at specific loci, without altering the underlying DNA sequence. g Chromosomal imaging: dCas9 is used in conjunction with guide RNAs bearing PP7 hairpins and fluorescent proteins (for example, GFP-MCP) to visualize specific genomic loci in live cells.(Figure created using Biorender.com).
