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A high-throughput; streamlined cloning protocol to generate guide RNAs for CRISPR activation
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DOI：10.3389/fgeed.2026.1787203.png)
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Abstract
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Caenorhabditis elegans is a powerful model for studying gene function and disease pathogenesis. While RNA interference effectively suppresses gene expression; CRISPR activation (CRISPRa) provides a robust tool for upregulating endogenous gene expression in C. elegans. Compared with traditional injection-based methods; feeding-based CRISPRa; similar to RNA interference; is easy; cost-effective and efficient. However; the traditional cloning workflows remain a bottleneck for high-throughput gene screening. Here; we present a pooled; one-step dual gRNA cloning protocol that enables rapid and efficient construction of gRNA expression vectors for CRISPRa. In a test case; by designing 55-mer and 54-mer primers; each containing one gRNA; we amplified and pooled 126 gRNA inserts in a single reaction pipeline. The 126 gRNA clones were completed in three pooled rounds; achieving 42%–56% coverage for each round; with remaining clones processed individually. This protocol dramatically reduces time; labor; and reagent consumption; while increasing scalability and maintaining reproducibility. It is particularly well suited for high-throughput screening of gene libraries or pathways and supports downstream applications such as phenotypic screening and lifespan analysis. This work advances CRISPRa-based functional genomics in C. elegans by providing a practical tool for large-scale gene activation studies.
Keywords:
high-throughput screening
C. elegans
cloning
CRISPR activation
guide RNA
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