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bioRxiv · 10.1101/2025.02.16.638485

CRISPR-based shuttle cloning of 1,397 human genes into UAS vectors

Liu, X.·Xi, H.·Dai, M.·Li, X.·Xue, W.·Chen, G.·Yan, J.·Xu, S.·Ou, G.·Luo, S.·Tang, Y.·Wei, P.·Wang, J.
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Abstract

The construction of genome-wide DNA libraries from publicly available resources is essential for leveraging functional genomics to investigate complex biological systems. However, all existing high-throughput cloning methods for transferring DNA fragments between vectors require PCR amplification of the DNA fragments, rendering the construction of genome-wide DNA libraries labor-intensive and time-consuming. By introducing a concept of CRISPRshuttle cassette, we herein present a method named CRISPR-based shuttle cloning (CRISPRshuttle cloning). This method enables the high-throughput transfer of numerous DNA fragments from original plasmids with identical backbones to a different vector background without the need for PCR amplification of the DNA fragments. The procedure comprises two-step test tube reactions followed by bacterial transformation. Using CRISPRshuttle we successfully generated a library of GAL4/UAS-based UAS-ORF plasmids covering 1,397 human genes conserved in Drosophila. This library may serve as a valuable resource for gain-of-function screening in cultured cells and for the creation of a transgenic UAS-ORF library in Drosophila.

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Liu, X., Xi, H., Dai, M., Li, X., Xue, W., Chen, G., Yan, J., Xu, S., Ou, G., Luo, S., Tang, Y., Wei, P., Wang, J.. 2025-02-16. CRISPR-based shuttle cloning of 1,397 human genes into UAS vectors. https://doi.org/10.1101/2025.02.16.638485

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