Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila

• Published in: Genetics (Austin) Vol. 227; no. 4
• Main Authors: Nyberg, Kevin G, Navales, Fritz Gerald, Keles, Eren, Nguyen, Joseph Q, Hertz, Laura M, Carthew, Richard W
• Format: Journal Article
• Language: English
• Published: US Oxford University Press 07.08.2024; Genetics Society of America
• Subjects: Alternative splicing; Cleavage; CRISPR; Drosophila; Fluorescence in situ hybridization; Fruit flies; Gene expression; Gene sequencing; Genes; Genetic engineering; Insects; Non-coding RNA; Nucleotide sequence; Nucleotides; Parasites; Ribonucleic acid; Ribozymes; RNA; RNA editing; Robustness; splice variants; knockdown; noncoding RNAs; gene expression; ribozyme; Ribozyme
• ISSN: 1943-2631; 0016-6731; 1943-2631
• DOI: 10.1093/genetics/iyae067

Abstract The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3′ fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes. Techniques for genetic manipulation in Drosophila melanogaster are powerful but not without limitations. Here, the authors detail a novel approach to achieve strong RNA knockdown in Drosophila using self-cleaving ribozymes. Ribozyme insertion via CRISPR/Cas9 into both nuclear and cytoplasmic noncoding RNAs and a male-specific splice variant of doublesex resulted in cleavage and efficient degradation of 3’ RNA fragments without observable off-target effects. This work demonstrates the potential of self-cleaving ribozymes for gene knockdown in Drosophila.