GLOSSARY OF FUSION PROTEINS AND TOOLS


SUMMARY

CRISPR-cas gene editors can be utilized directly for their nuclease activity, but many emerging alternative strategies for genome modification utilize a cas effector fused to an additonal protein(s). The RNA-guided targeting capabilities guide the Cas fusion to the desired edit location, and the fused protein edits the targeted locus. Fusion proteins can not only alter the DNA sequence, but also regulate transcription and translation by recruiting gene expression machinery or adding DNA and histone modifications. These CRISPR-cas gene editor fusions can offer several advantages over the CRISPR nuclease in certain therapeutic applications, and several of these fusions have moved rapidly into the clinic. Extensive engineering efforts 1 have improved these fusions editing capablities.

TYPES OF FUSIONS

Fusion Name Fusion Description dsDNA Nuclease, Nickase, or dCas? Representative Fusion Partners
CRISPR interference (CRISPRi) CRISPRi utilizes a deactivated Cas9 (dCas9) protein fused with transcriptional repressors (e.g. KRAB) to inhibit gene expression, allowing precise control over gene function and investigation of gene regulatory mechanisms. 2 dCas KRAB, ZIM3 KRAB,DLN144, DLS, MIX
CRISPR activation (CRISPRa) /CRISPR-on CRISPRa employs a modified Cas9 protein (dCas9) fused with transcriptional activators to enhance gene expression, enabling targeted upregulation of specific genes for functional studies and therapeutic applications. 2 dCas VP16, VP64, Rta (VPR), SunTag (GCN4-VP64)
CRISPRoff CRISPRoff is a programmable epigenetic memory writer composed of a deactivated Cas9 (dCas9) fused to chromatin modifiers that establish DNA methylation and repressive histone modifications. 3 dCas ZNF10 KRAB, Dnmt3A (D3A), Dnmt3L (D3L)
CRISPRon CRISPRon is a programmable epigenetic memory modifier which both removed DNA methylation and recruits transcriptional machinery 3. The same term, CRISPR-on has been used to describe transient transcriptional activation as well, which is detailed under CRISPRa/CRISPR-on 4. dCas VP64, p65-AD, and Rta
Adenine Base Editors (ABE) Adenine base editors refer to a deactivated Cas9 (dCas9) fused to an adenine base editor, typiclly an engineered TadA deaminase. This fusion is capable of making targeted base transitions from A to G (or T to C on the opposite stand). 5 6. To expand this technology to targeted base transversions (A to C/T), Cas9 nickase is fused to TadA along with and alkyladenine DNA glycosylase 7. nCas (Cas9 D10A) or dCas TadA, mAAG
Cytosine Base Editors (CBE) Cytosine base editors refer to a deactivated Cas9 (dCas9) fused to a cytosine deaminase. This fusion is capable of making targeted base transitions from C to T (or G to A on the opposite strand). 5. To prevent cytosine transversion side products, Cas9 nickase can be fused to cytosine deaminase along with a uracil glycosylase inhibitor; fidelity is further improved by a fusion with Gam from bacterphage Mu 8. To expand this technology to targeted base transversions (C to G), Cas9 nickase can be fused to a uracil DNA N-glycosylase (eUNG) and a cytodine deaminase (APOBEC1) 9. nCas (Cas9 D10A) or dCas APOBEC3A, CDA, APOBEC1, AID, AIDx with or without UGI and Mu Gam
RNA Deaminases In these fusion systems, sometimes referred to as REPAIR (RNA editing for programmable A to I (G) replacement) and RESCUE (RNA Editing for Specific C to U Exchange) or CURE (C to U RNA Editor), deaminases that act on double stranded RNA are fused to deactivated Cas13b to enable targeted base modifications from C to T or A to G. 10 dCas ADAR1, ADAR2, APOBEC3A
Prime Editors Prime editors refer to a modifed nickase Cas9 fused to a reverse transcriptase. This fusion, guided by a prime editing guide (pegRNA), is able to make targeted deletions, insertions and point mutations; the pegRNA consists of a gRNA sequence at the 5' end and a primer binding site + template for reverse transcription at the 3' end, the product of which will be incorporated into the nicked strand as edited DNA 11 nCas (Cas9 H840A) MMLV, MMLVΔRH
Recombinase The RecCas9 system employs a Cas-recombinase fusion to recombine DNA sites flanked by guide RNA-specified sequences 12 dCas Gin
Transposase These fusions, CasTn, employ DNA insertion mechanisms from transposons to direct the targeted integration of DNA into the genome. 13,14 dCas, nCas Himar1,TnpA
Integrase Programmable addition via site-specific targeting elements (PASTE), uses a fusion protein of Cas9 nickase with a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of large DNA sequences. 15 nCas Bxb1, TP901, phiBT1
APEX2 Proximity Labeling By fusing catalytically-dead Cas systems with the engineered peroxidase APEX2, proteins proximal to a gRNA-defined DNA or RNA locus can be labeled with Biotin, thus identifying putative protein-nucleotide interaction partners. Cas13d-based 16 and Cas9-based 17 systems have been developed to identify RNA and DNA interaction partners, respectively. dCas APEX2

TOOLS

Enzyme CRISPRi CRISPRa/CRISPR-on CRISPRoff CRISPRon Adenine Base Editors (ABE) Cytosine Base Editors (CBE) RNA Deaminases Prime Editors Recombinases Transposases Integrases APEX2
SpyCas9 Tool Exists18,19,2,20 Tool Exists2,19,21 Tool Exists3 Tool Exists3 Tool Exists5,7 Tool Exists5,8,9 Tool Exists11,22,23 Tool Exists12 Tool Exists13,14 Tool Exists15 Tool Exists24,17
SauCas9 Tool Exists25,26 Tool Exists26 Tool Exists27,28,29 Tool Exists30,31 Tool Exists32
AsCas12a Tool Exists26 Tool Exists33 Tool Exists27 Tool Exists33,34
LbCas12a Tool Exists26 Tool Exists26 Tool Exists35 Tool Exists35,34,36
Un1Cas12f Tool Exists37 Tool Exists37Tool Exists38
Nme2Cas9 Tool Exists39
ScCas9 Tool Exists40 Tool Exists40,41
SauriCas9 Tool Exists42 Tool Exists42
CjeCas9 Tool Exists43 Tool Exists43
CWCas12f Tool Exists38
FnCas9 Tool Exists44
GBSCas9
DpbCas12e Tool Exists26,45 Tool Exists26,45
PlmCas12e Tool Exists26 Tool Exists26
Cas12j2/3 Tool Exists26,46 Tool Exists26,46
Cas13b Tool Exists47,48,49
HEARO Tool Exists50
Cas9d Tool Exists50 Tool Exists50
Cas13d Tool Exists16

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