Engineered bacteria detect tumor DNA

Abstract

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Figure 1.. Engineered bacteria to detect tumor DNA.

Engineered A. baylyi bacteria are delivered rectally in an orthotopic mouse model of CRC. The naturally competent A. baylyi take up tumor DNA shed into the colorectal lumen. The tumor donor DNA is engineered with a kanR cassette flanked by KRAS homology arms. The sensor bacteria are engineered with matching KRAS homology arms that promote homologous recombination. Sensor bacteria that undergo HGT from tumor DNA acquire kanamycin resistance and are quantified from luminal contents by serial dilution on antibiotic selection plates.

Figure 2:. Sensing KRASG12D DNA in vitro.

A, Donor DNA was derived from plasmid, purified cancer cell genomic DNA, or raw lysate (top) that recombined into biosensor A. baylyi cells (bottom). Horizontal gene transfer included either a large, 2 kb insert B, or a small, 8 bp insert to repair 2 stop codons C, in both cases conferring kanamycin resistance. D-G) A. baylyi biosensors were incubated with plasmid DNA, purified RKO-KRAS or LS174T-KRAS genomic DNA, or raw RKO-KRAS lysate, all containing the donor cassette, or purified RKO or LS174T genomic DNA as controls. Biosensor cells included either “large insert” (B, D & E) or “small insert” (C, F & G) designs, and transformations were performed in liquid culture (D & F) or on solid agar surfaces (E & G). Two sample t-tests compared data to RKO and LS174T genomic DNA controls under the same conditions. H, CRISPR spacers targeting the KRAS G12D mutation (boxed), using the underlined PAMs. Fraction of total biosensor cells expressing the indicated CRISPR spacers that were transformed by plasmid donor DNA with wild type (I) or mutant G12D (J) KRAS. Statistics were obtained using two sample, one-sided t-tests, with p-values displayed on the figures. Data points below detection are shown along the x-axis, at the limit of detection.

Figure 3:. Detection of donor DNA from BTRZI-KRAS-kanR organoids in both an in vitro and an in vivo model of colorectal cancer.

A, Schema depicting in vitro co-culture of A. baylyi sensor bacteria with BTRZI-KRAS-kanR (CRC donor) organoid lysates or viable organoids to assess HGT repair of kanamycin resistance gene (kanR). B, Recombination with DNA from crude lysates enables growth of A. baylyi sensor on kanamycin. C, Representative images of GFP-tagged A. baylyi biosensor surrounding parental BTRZI (control) and BTRZI-KRAS-kanR donor organoids at 24h. Scale bar 100 μm. D, Co-culture of established CRC BTRZI-KRAS-kanR donor organoids with A. baylyi sensor enables growth of A. baylyi sensor on kanamycin. In B & D, n = 5 independent experiments each with 5 technical replicates, one sample t-test on transformed data was used for statistical analysis with p-values as indicated. E, Schema depicting in vivo HGT experiments: generation of BTRZI-KRAS-kanR (CRC donor) tumors in mice via colonoscopic injection, with tumor pathology validated by H&E histology, administration of biosensors, and analysis of luminal contents. Scale bars 200μm. F, rectal delivery of A. baylyi biosensor to mice bearing CRC donor tumors results in kanamycin resistant A. baylyi biosensor in luminal contents via HGT with transformation efficiency of 1.5×10⁻⁹ (limit of detection 1.25×10⁻¹⁰). HGT rate calculated from CFU on kanamycin/chloramphenicol/vancomycin (transformants) and chloramphenicol/vancomycin (total A. baylyi) selection plates, n=3–5 mice/group. One-way ANOVA with Tukey’s post-hoc on log10 transformed data was used for statistical analysis. G, ROC curve analysis of HGT CFU following enema, AUC = 1, p = 0.009.

Figure 4:. Detection of non-engineered DNA.

A, tetR located between the homology arms on the A. baylyi genome represses expression of the output gene. B, Target DNA with wild-type KRAS sequence is recognized and degraded by the Type I-F CRISPR-Cas effector complex, Cascade. C, Target DNA with the KRASG12D mutation avoids degradation, replaces tetR in the biosensor genome, and relieves repression of the output gene. Fraction of biosensors with either a random CRISPR spacer D, or a spacer targeting wild type KRAS E, that detected donor DNA. Statistics were obtained via two sample t-tests and are displayed on the figure.