Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium

Abstract

Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.

Keywords: CRISPR-Cas; bifidobacterium; epigenomics; genomics; probiotics.

Fig. 1.

CRISPR-Cas systems transcription profiles based on RNA-seq data. (A) Transcriptional profile of the type I-G CRISPR locus in B. lactis DSM 10140, with mRNA in blue and small RNA (smRNA) in yellow. (B) Mature type I-G crRNA determined by smRNA sequencing (smRNA-seq). The pre-crRNA processing sites are indicated by black arrows. The spacer sequence is highlighted in red and the repeat sequence is underlined, with the cleaved sequence in gray and retained sequence in black. (C) Transcriptional profile of the type II-C CRISPR locus in B. longum DJO10A. (D) Mature type II-C crRNA and tracrRNA determined by smRNA-seq, along with the predicted crRNA:tracrRNA duplex.

Fig. 2.

Functionality of endogenous CRISPR-Cas systems in Bifidobacterium. (A) A schematic diagram of CRISPR-Cas system characterization using a cell-free transcription-translation (TXTL) system. (B, C) Characterization of type I-G and type I-E functionality in TXTL, respectively. The relative fluorescence reported in the bar graphs is calculated by dividing the background-corrected fluorescence of targeting spacer by the nontargeting spacer at the 16 h end point. (D, E) Plasmid interference assay of type I-G and type I-E system in Bifidobacterium, respectively. (F) Sanger sequencing revealed that the surviving colonies in Bi-26 (type I-E) transformation with 5′-GAAG-3′ PAM carried mutations in the PAM sequence but not in the negative control associated with a 5′-ATGT-3′ PAM. Data shown in the bar graphs represents the average of three independent biological replicates (except for E where two biological replicates were performed), with the SD displayed as error bars. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on Welch’s t test to compare the sample average with the negative control.

Fig. 3.

Repurposing the endogenous type I-G CRISPR-Cas system to generate large deletion events in Bifidobacterium. (A) The DSM 10140 genome was mapped with predicted essential genes (black), insertion sequence (IS) (red), and predicted islands (gray). (B) The CRISPR-based large deletion screening plasmid, a pBC1-based shuttle vector expressing a CRISPR array driven by the native leader. (C) Plasmid transformation efficiencies. **P < 0.01, based on Welch’s t test to compare the self-targeting average transformation efficiency with the random control. (D) Genome sequencing revealed that by targeting lacI and lacA using the endogenous type I-G system, large deletion events (27 kb and 25 kb, respectively) were observed. (E) PCR amplicons generated from combinations of internal and flanking primers. (F) Schematic overview of the recombinogenic deletion events.

Fig. 4.

Repurposing the endogenous type I-G CRISPR-Cas system for genome editing in Bifidobacterium. (A) Plasmid pTRK1278, a pBC1-based E. coli–Bifidobacterium shuttle vector, was used to generate CRISPR-based genome editing in Bifidobacterium. (B) Schematic overview of a 500-bp-deletion containing the promoter region, start codon and the 5′ portion of tetW. The CRISPR array expresses a spacer matching the 5′ end of tetW (within the deletion region, indicated in red), flanked by a 5′-TAT-3′ PAM. (C) Plasmid transformation efficiencies. **P < 0.01, ***P < 0.001 based on Welch’s t test to compare each plasmid transformation efficiency with the positive control pTRK1278. (D) Chromatogram profiles demonstrating the 500-bp-deletion was achieved in B. lactis DSM 10140 by repurposing its native type I-G CRISPR-Cas system. (E) PCR amplicons generated from flanking primers revealed a 500-bp-deletion in the ΔtetW mutant NCK2931 compared to the wild type. (F) The minimal inhibitory concentration (MIC) test demonstrated that the 500-bp-deletion rendered the mutant sensitive to tetracycline.

Fig. 5.

Application of a portable CRISPR-Cas cytosine base editor for genome editing in Bifidobacterium. (A) The nCas9-APOBEC-1 fusion along with a sgRNA was cloned into pTRK1278, generating the base editing plasmid pTRK1284. (B) Schematic overview of the cytosine base editor introducing C•G-to-T•A mutation in tetW in B. lactis DSM 10140. The sgRNA targets a protospacer flanked by a 5′-NGG-3′ PAM at the 5′ end of tetW, targeting the fifth nucleotide within the protospacer. (C) MIC testing revealed that the p.Gln228Ter nonsense mutation rendered the mutant NCK2932 sensitive to tetracycline. (D) The portable base editor generated C•G-to-T•A mutation in B. lactis strains with various efficiencies. (E) Alignment of the uracil-DNA glycosylase coding sequence (udg) from all five B. lactis strains revealed a c.351_delinsC insertion in DSM 10140.

Fig. 6.

Epigenomic mapping of Bifidobacterium to assess genomic context variability. (A) The expected motifs of B. lactis and B. infantis, as well as a de novo 5mC motif that is shared between the species, are shown with neural network (NN) scoring using Nanodisco. (B) The heatmap shows the NN scoring prediction for each motif in the various strains. Red boxes indicate the fine mapping locations in the motifs. White boxes indicate the motif was detected de novo with MEME, but was below NN prediction scoring, while gray boxes show the motif was not detected de novo with MEME. (C) The metagenomic binning scores across the genomes of all strains are compared using one-way ANOVA with DSM 10140 as the control (**P < 0.05, ***P < 0.001, ****P < 0.0001). (D) Three methylation motifs 5′-GGW5mCC-3′ (gray), 5′-RTC6mAGG-3′ (red), and 5′-5mCACC-3′ (blue) were mapped across (D) the lac islands in B. lactis DSM 10140, (E) the tetW and IS5-like element region in DSM 10140, and (F) plasmid pTRK1278 using Geneious Prime. Green regions show transposase sequence for lacI island, and homologous sequences for lacA island. The deletion repair templates for the tetW region are shown below the gold gene annotations as blue arrows for base editing and green arrows for endogenous CRISPR-Cas targets.