Related Experiment Video
Updated: Jan 18, 2026
11:53
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
Published on: July 12, 2017
19.0K
Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs
Fillip Port1,2,3, Martha A Buhmann4,5,6,7, Jun Zhou4,5,6,8
1Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany. f.port@dkfz.de.
Nature Communications
|January 15, 2026
Summary
This study introduces a new CRISPR Cas12a system using multiple guide RNAs in Drosophila for precise gene knock-out. This multiplexed approach enhances efficiency and accuracy, overcoming limitations of previous gene editing tools.
Area of Science:
- Molecular Biology
- Genetics
- Gene Editing Technologies
Background:
- CRISPR gene knock-out faces challenges including inefficient single guide RNAs (sgRNAs), inaccessible DNA targets, and unpredictable DNA repair.
- Existing Cas9 systems have limitations in efficiency and specificity for comprehensive gene disruption.
Purpose of the Study:
- To develop and validate a novel Cas12a-based CRISPR system in Drosophila utilizing multiplexed sgRNAs to enhance gene knock-out efficiency and reliability.
- To assess the on-target and off-target activity of this multiplexed Cas12a system across a significant portion of the Drosophila genome.
Main Methods:
- A multiplexed CRISPR system employing the Cas12a nuclease and four single guide RNAs (sgRNAs) per target gene was designed for Drosophila.
- A high-throughput screening assay was developed to visualize and quantify CRISPR-nuclease activity in living animals.
- The system's performance was evaluated across 33% of the Drosophila genome using over 2000 sgRNAs and compared directly against established Cas9-based methods.
Main Results:
- Multiplexed sgRNAs demonstrated synergistic effects, leading to increased deletion formation between target sites and a higher frequency of loss-of-function mutations.
- The system exhibited high on-target activity (>99%) and very low off-target activity (<1%) in genome-wide screening.
- Multiplexed Cas12a gene targeting outperformed Cas9-based systems in parallel gene targeting experiments, revealing previously undetected phenotypes.
Conclusions:
- The multiplexed Cas12a system provides a robust and efficient method for reliable gene knock-out in multicellular organisms like Drosophila.
- This advanced gene editing framework overcomes key limitations of current CRISPR technologies, enabling more comprehensive genetic analysis and discovery.
