Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

G3, 2021 · DOI: 10.1093/g3journal/jkab089 · Published: March 20, 2021

Neurology Genetics Regenerative Medicine & Stem Cells

Simple Explanation

This study introduces a method for efficiently modifying genes in adult zebrafish using CRISPR/Cas9 technology, combined with behavioral tests to identify genes involved in spinal cord repair. The researchers targeted 17 genes and found that they could effectively disrupt these genes in adult fish. They then used a swimming test to see how well the fish recovered after a spinal cord injury, identifying several genes that seem to be important for this process. To confirm their findings, they created stable genetic mutations in some of these genes and observed similar effects on spinal cord regeneration, suggesting that this approach can be used for large-scale genetic studies in adult zebrafish.

Study Duration

4 Weeks

Participants

Adult zebrafish (Tubingen strain), both male and female, between 3 and 6 months old, ~2 cm in length

Evidence Level

Experimental study using CRISPR/Cas9 mutagenesis and behavioral assays

Key Findings

1
CRISPR/Cas9 dual-guide ribonucleic proteins (dgRNPs) can achieve selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 crispants.
2
Seven single- or duplicate-gene crispants showed reduced functional recovery after spinal cord injury, as identified using a quantifiable behavioral assay.
3
Germline mutations generated to rule out off-target effects recapitulated the crispant regeneration phenotypes, validating the role of the targeted genes in spinal cord repair.

Research Summary

The study combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Seventeen genes were targeted, and the resulting mutants were screened for functional recovery after spinal cord injury, revealing several genes crucial for this process. The findings were validated by generating germline mutations that phenocopy the results seen in the initial CRISPR/Cas9-based screen.

Practical Implications

Drug Target Identification

The identified genes can be further investigated as potential therapeutic targets for promoting spinal cord regeneration in humans.

Advancement of Genetic Screening

The developed pipeline can be applied to study other biological processes and diseases in adult zebrafish, expanding the scope of genetic research.

Refinement of Genome Editing Techniques

The study provides insights into optimizing CRISPR/Cas9-based mutagenesis for efficient and specific gene targeting in adult organisms.

Study Limitations

1
The potential for off-target effects caused by CRISPR/Cas9 dgRNPs cannot be completely ruled out.
2
Small in-frame alleles were generated at various frequencies, which may introduce phenotypic noise and complicate the interpretation of certain phenotypes.
3
The inability to achieve temporal or spatial specificity in adult tissues with dgRNP-based mutagenesis limits the study of gene function in specific contexts.

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