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High-throughput retrieval of target sequences from complex clone libraries using CRISPRi

Nature Biotechnology volume 41, pages 626–630 (2023)Cite this article

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Abstract

The capture of metagenomic DNA in large clone libraries provides the opportunity to study microbial diversity that is inaccessible using culture-dependent methods. In this study, we harnessed nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC) that can be used to retrieve target clones from complex libraries. Combining modern sequencing methods with CCIC cloning allows for rapid physical access to the genetic diversity present in natural ecosystems.

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Fig. 1: CCIC development and its application for target sequence retrieval.
Fig. 2: Targeted retrieval from metagenomic and genomic CCIC libraries.

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Data availability

pCCIC has been deposited as GenBank ON804120. Recovered BGC clusters containing cosmid clones have been deposited as GenBank ON996267–ON996333 and OP058960. The S. albidoflavus J1074 reference genome used is publicly available as GenBank CP004370. The Comprehensive Antibiotic Resistance Database, used for antibiotic resistance analysis of sucrose escape clones, is publically available at https://card.mcmaster.ca. Other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

Publicly available Flye version 2.9-b1768, antiSMASH 5.1, Defense Finder 1.0.8, SeqKit version 2.1.0, VSEARCH version 2.18.0, minimap2 version 2.24-r1122 and UGENE version 42.0 were used for sequence assembly and analysis. Custom code generated for edge mapping has been deposited in Zenodo (https://doi.org/10.5281/zenodo.6574918).

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Acknowledgements

This work was supported by National Institutes of Health grant 5R35GM122559 (S.F.B.). We thank the Marraffini laboratory for pdCas9. Vectors pCas (Addgene, 62225) and pTargetF (Addgene, 62226) were gifts from Sheng Yang, and pwtCas9-bacteria (Addgene, 44250) was a gift from Stanley Qi. PacBio sequencing was performed by the Rockefeller University Vertebrate Genome Center.

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Authors and Affiliations

  1. Laboratory of Genetically Encoded Small Molecules, Rockefeller University, New York, NY, USA

    Ján Burian, Vincent K. Libis, Yozen A. Hernandez, Liliana Guerrero-Porras, Melinda A. Ternei & Sean F. Brady

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  1. Ján Burian
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Contributions

J.B., V.K.L. and S.F.B. conceived CCIC retrieval. J.B. and S.F.B. designed and analyzed experiments. J.B. performed all experiments, with the aid of M.A.T. for metagenomic DNA preparation and lambda packaging, L.G. for various cloning and Y.A.H. for bioinformatics.

Corresponding author

Correspondence to Sean F. Brady.

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Competing interests

S.F.B. has consulted for Zymergen. All other authors declare no competing interests.

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Nature Biotechnology thanks Benjamin Rubin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Methods, Supplementary Figs. 1–7 and Supplementary Notes 1–3

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Supplementary Tables 1–6

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Burian, J., Libis, V.K., Hernandez, Y.A. et al. High-throughput retrieval of target sequences from complex clone libraries using CRISPRi. Nat Biotechnol 41, 626–630 (2023). https://doi.org/10.1038/s41587-022-01531-8

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