Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs

Nature Communications 2026 Open access

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Authors: Fillip Port, Martha A Buhmann, Jun Zhou, Mona Stricker, Alexander Vaughan-Brown, Ann-Christin Michalsen, Eva Roßmanith, Amélie Pöltl, Lena Großkurth, J. Huber, Laura B Menendez Kury, Bea Weberbauer
+4 more Maria Hübl, Elli Puscher, Florian Heigwer, Michael Boutros
DOI: 10.1038/s41467-026-68434-z
Cited by: 4
References: 56

Abstract

CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (

Fields of Study & Subjects

CRISPR and Genetic Engineering Pluripotent Stem Cells Research RNA Interference and Gene Delivery

Publication details

Journal / source
Nature Communications
Publisher
Springer Science and Business Media LLC
Volume
17
Issue
1
Pages
877–877
Publication date
15 January 2026
Language
EN