biostudies-literatureShah KMBiotechnology and Biological Sciences Research Council1-24https://www.ebi.ac.uk/biostudies/studies/S-EPMC12296108biostudies-literatureUnknown22(1)MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the <i>N</i>- and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous CRNA biologyC-terminal tagging impairs AGO2 function.PMC12296108BB/V009567/1Sharp TVShah KMCrozier AFFAssaraf APlevin MJMardakheh FGrevitt PArya MfalseC-terminal tagging impairs AGO2 function.MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the <i>N</i>- and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous C2025-01-01T00:00:00Z2025 Dec2026-03-15T18:40:32.596Z2025-08-13T03:04:36.945ZS-EPMC122961084069864510.1080/15476286.2025.2534028