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Simultaneous multi-site editing of individual genomes using retron arrays

Abstract

During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology—termed a multitron—for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.

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Fig. 1: Encoding several donors in a retron msd enables multiplexed retron recombineering.
Fig. 2: Improved multiplexed editing using donors in arrayed retron msds.
Fig. 3: Increasing limits of deletion size using nested deletion donor arrays.
Fig. 4: Multi-site editing of individual bacterial genomes using multitrons.
Fig. 5: Metabolic engineering using multitrons.
Fig. 6: Arrayed retron msds enable multiplexed editing in eukaryotic cells.

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Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information or will be made available from the authors upon request. Sequencing data associated with this study are available on NCBI SRA as BioProject ID PRJNA1107632.

Code availability

Custom code to process or analyze data from this study is available via GitHub at https://github.com/Shipman-Lab/multitrons (https://doi.org/10.5281/zenodo.11289190).

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Acknowledgements

Work was supported by funding from the National Science Foundation (MCB 2137692), the National Institute of Biomedical Imaging and Bioengineering (R21EB031393), the National Institute of General Medical Sciences (1DP2GM140917) and the UCSF Program for Breakthrough Biomedical Research. S.L.S. is a Chan Zuckerberg Biohub—San Francisco investigator and acknowledges additional funding support from the L.K. Whittier Foundation and the Pew Biomedical Scholars Program. A.G.-D. was supported by the California Institute of Regenerative Medicine (CIRM) scholar program. S.C.L. was supported by a Berkeley Fellowship for Graduate Study.

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S.L.S., A.G.-D. and S.C.L. conceived the study. A.G.-D., S.C.L. and M.R.-M. cloned plasmids used in this study, A.G.-D. performed all experiments with E. coli, and S.C.L. performed all experiments with S. cerevisiae and human cultured cells. M.R.M. and C.B.F. performed NGS and prepped and ran the sequencing libraries. A.G.-D., S.C.L. and S.L.S. designed the work, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Seth L. Shipman.

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Competing interests

A.G.-D., S.C.L. and S.L.S. are named inventors on a patent application related to the technologies described in this work (63/524,317). S.L.S. is a founder of Retronix Bio. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Trans msr multitron architecture enables precise genome editing.

Top: Schematic of retron recombineering using an msd array with a single msr sequence in trans including a terminator (T) between the msd array and msr. Bottom: quantification of precise editing rates for precise editing of rpoB or gyrA simultaneously by Illumina sequencing after 24 h of editing. Circles show each of the three biological replicates, bars are mean ± SD.

Extended Data Fig. 2 Optimization of retron recombineering using a single plasmid.

a. Left: schematic of the different retron operon architectures tested. ncRNA with donor (orange and blue), genes required (grey) and optimized ribosome binding sites (RBS) regions (green) are indicated Right: quantification of rates for precise rpoB editing, circles show each of the three biological replicates, bars are mean ± SD. b. Quantification of precise editing rates for rpoB target site at 30 and 37 °C, circles show each of the three biological replicates, lines are mean ± SD. c. Quantification of OD600 using increasing concentrations of m-toluic acid after 16 h of bacterial growing (n = 1). d. Quantification of precise editing rates for rpoB using different concentrations of arabinose (n = 1). e. Quantification of colonies with intact msd arrays. A total of 30 colonies coming from 3 different replicates were sequenced, bars are mean ± SD All precise editing rates were quantified using Illumina MiSeq after 24 h of editing. f. Scheme of the protocol used to analyze genetic stability of the retron arrays. Briefly, recombineering plasmid was transformed into E. coli strain bMS.346, followed by 5 days of growing and diluting in the presence or absence of the arabinose. A dilution of the final culture was diluted and plated. Finally, the msd Array of 10 individual colonies per replicate (n = 3) were amplified and sequenced to assess genetic stability of the multitron approach.

Extended Data Fig. 3 Local off-target mutations.

a. Quantification of precise editing rates for fbaH and hda genes using a live or dead version of Eco1 RT, circles show each of the three biological replicates, bars are mean ± SD. b. Local off-target mutation frequency in the 70 bp region of the chromosome homologous to fbaH and hda editing donors using a live of dead version of Eco1 RT circles show each of the three biological replicates, bars are mean ± SD. All data was quantified using Illumina MiSeq after 24 h of editing.

Extended Data Fig. 4 Intended and undesired on-target mutation rates caused by arrayed retron multiplexed editing in yeast cells.

a. Top: Schematic of the donor encoding retron ncRNA/gRNA expression cassette expressed from a Gal7 Pol II promoter and flanked by ribozymes versus a new construction replacing ribozymes with Csy4 sequences. Bottom left: schematic of a retron ncRNA-Cas9 gRNA hybrid for genome editing in yeast, depicted above the protein-coding expression cassette which is inserted into the yeast genome. Bottom right: quantification of indel rates of the ADE2 locus in yeast by Illumina sequencing after 48 h of editing. Circles show each of the three biological replicates, bars are mean ± SD; absence/presence of Csy4 in the protein-coding expression cassette is shown below the graph. b. Top: schematic of an arrayed retron ncRNA-Cas9 gRNA expression cassette, expressed from a Gal7 Pol II promoter, flanked by ribozymes, and separated by a Csy4 sequence. The retron editors in positions 1 and 2 target the ADE2 and FAA1 locus, respectively. Bottom: quantification of indel rates of the ADE2 and FAA1 loci in yeast by Illumina sequencing after 48 h of editing. Circles show each of the three biological replicates, bars are mean ± SD; absence/presence of Csy4 in the protein-coding expression cassette is shown below the graph. c. Top: schematic of an arrayed retron msdRNA-Cas9 gRNA expression cassette, expressed from a Gal7 Pol II promoter, flanked and separated by a Csy4 sequence; the msrRNA is expressed in trans from a SNR52 Pol III promoter. Bottom: assembly schematic for one-pot Golden Gate cloning of multiple msdRNA-sgRNA editors. d. Schematic showing the presumed processing, annealing and reverse-transcription involved in the generation of editing donors from arrayed retron msdRNA-Cas9 gRNA cassettes. e. top: schematic of 5x arrayed retron msdRNA-Cas9 gRNA expression cassettes, as shown in Extended Data Fig. 4c. Bottom: quantification of precise editing of the various yeast loci targeted by the retron editors shown above, by Illumina sequencing, after 24 and 120 h of editing. The editors target ADE2, CAN1, TRP2, SGS1 and FAA1. Two-way ANOVA, effect of expression time, P = 0.0038. Circles show each 3 biological replicates, bars are mean ± SD. f–h, top: schematic of 2x, 3x or 5x arrayed retron msdRNA-Cas9 gRNA expression cassettes. Bottom: quantification of indel rates of the various yeast loci targeted by the retron editors shown above, by Illumina sequencing, after 24 and 120 h of editing. Individual open circles show each of three biological replicates per condition, bars are mean ± SD The editors target ADE2 and FAA1 (f); ADE2, CAN1 and FAA1 (g); and ADE2, CAN1, TRP2, SGS1 and FAA1 (h).

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González-Delgado, A., Lopez, S.C., Rojas-Montero, M. et al. Simultaneous multi-site editing of individual genomes using retron arrays. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01665-7

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