Precision targeting of genetic variations in mixed bacterial cultures using CRISPR-Cas12a-programmed λ phages

Abstract

The CRISPR-Cas system, an adaptive immune mechanism in prokaryotes against bacteriophages, has been developed into a versatile tool for recognizing and cleaving target nucleic acid sequences. In this study, we developed a model system by integrating CRISPR-Cas12a into the genome of temperate bacteriophage λ, enabling precise regulation of lysogeny and lysis in Escherichia coli. We confirmed that λ phage, armed with Cas12a nuclease and CRISPR RNA (crRNA) targeting specific sequences, could inhibit the lysogenic cycle of E. coli cells. We demonstrated that the CRISPR-Cas12a-loaded temperate λ phage mimicked a lytic phage by selectively killing cells carrying the target genomic sequence. Furthermore, by employing truncated crRNA to enhance target recognition specificity, we found that the synthetic phage could distinguish single nucleotide variations in the genomic target DNA, enabling precise targeting and selective elimination of target cells in homogeneous bacterial cultures. To further validate its specificity, we tested this system in mixed bacterial cultures, wherein Cas12a nuclease and truncated crRNA-loaded bacteriophages selectively eliminated only those cells carrying the target sequences perfectly matching the crRNA. These results highlight the potential of this approach for advancing precision microbiome modulation.

Keywords: CRISPR-Cas12a; Escherichia coli; bacteriophage; lambda; lysogen.

Figure 1

Death of target DNA-containing cells induced by λ phage carrying Cas12a. (A) Construction of λ cas12a phage. The cas12a gene was inserted into the b2 region of the λ cI⁸⁵⁷ prophage genome via homologous recombination. (B) Spotting assay of various synthetic λ phages on galK mutant strains. λ cI⁸⁵⁷, λ Δb2, λ cI^antisense, and λ cas12a were spotted on LB top agar plates with ΔgalK or galK WT cells harboring galK-targeting crRNA plasmids. The plates were incubated at 30°C for 18 h. (C) Mechanism of cell death induced by λ cas12a. λ cas12a expresses the Cas12a nuclease and the crRNA plasmid in the host cell generates crRNA targeting the galK gene. This leads to the formation of the Cas12a–crRNA complex. If the host genome lacks the galK target, the phage remains in the prophage state without inducing cell death. Conversely, the presence of the galK target results in cell death.

Figure 2

Discrimination of single-nucleotide variations using Cas12a and truncated crRNA. (A) Spotting assay of λ cas12a on various galK strains at 30°C. N₂₃ and N₁₇-N₁₅ represent the length of the target recognition sequence (TRS) in the crRNA, and Δ in the parentheses indicates the number of truncated nucleotides at the 3′-end of the crRNA. (B) Mismatch intolerance. “504” represents the 504th nucleotide position in the galK gene of the E. coli genome. The nucleotides A and T in red represent the variant and original sequences, respectively. The black lines indicate base pairing. The scissors icon and red, dashed line show the position where the Cas12a–crRNA complex recognizes and cleaves the target DNA sequence. The red I indicates that the Cas12a–crRNA complex fails to recognize and cleave the target DNA.

Figure 3

Sequence-specific target recognition and cell death control using λ phage carrying Cas12a and crRNA. (A) Construction of λ cas12a-crRNA phage. The cas12a gene and crRNA gene targeting the galK gene were inserted into the b2 region of the λ cI⁸⁵⁷ prophage genome. (B) Spotting assay of various synthetic λ phages including λ cas12a-crRNA at 30°C. λ cI⁸⁵⁷, λ Δb2, λ cI^antisense, λ cas12a, and λ cas12a-crRNA were spotted on LB top agar plates containing ΔgalK, galK ⁵⁰⁴A, or galK WT cells, and incubated at 30°C for 18 h. N₂₃-N₁₅ represent λ cas12a-crRNA with crRNA TRS lengths ranging from 23 nt (Δ0) to 15 nt (Δ8). (C) Growth curves of galK ⁵⁰⁴A and galK WT strains either uninfected or infected with λ cI⁸⁵⁷, λ cI^antisense, λ cas12a galK-N₂₃, and λ cas12a galK-N₁₆, respectively, at 30°C. The gray arrow indicates the time point of phage infection. OD_{600 nm} values represent the mean obtained from three independent cultures.

Figure 4

Control of cell death in mixed cell cultures with different galK phenotypes using λ phages carrying the CRISPR-Cas system and lacZ. (A) Construction of the λ cas12a-crRNA-lacZ phage. The cas12a gene, galK-targeting crRNA, and lacZ gene were inserted into the b2 region of the λ cI⁸⁵⁷ prophage genome. (B) Verification of cell death and lysogen formation in galK ⁵⁰⁴A ΔlacZ and galK WT ΔlacZ strains on MacConkey agar plates. galK ⁵⁰⁴A ΔlacZ and galK WT ΔlacZ cells were cultured to early exponential phase, mixed at equal ratio, and either left uninfected or were infected with the phage. After 19 h of incubation, the cultures were spread on MacConkey D-Gal and MacConkey Lac plates. On the MacConkey D-Gal plate, white and red colonies represent galK ⁵⁰⁴A ΔlacZ and galK WT ΔlacZ cells, respectively. On the MacConkey Lac plate, white and red colonies represent non-lysogenic and lysogenic cells, respectively. (C) Growth curves of mixed cultures containing galK ⁵⁰⁴A ΔlacZ and galK WT ΔlacZ strains, either uninfected or infected with various synthetic λ phages at 30°C. The gray arrows on the left indicate the time point of phage infection, whereas gray arrows on the right indicate the time point of spreading. Each OD_{600 nm} value represents the mean of values for three independent cultures. (D) Proportions of galK ⁵⁰⁴A ΔlacZ and galK WT ΔlacZ strains, as well as lysogenic cells, observed on MacConkey agar plates. The gray bars represent the total number of viable cells (white colonies + red colonies) observed on the plates. The green bars indicate the proportion of galK WT ΔlacZ cells among the viable cells, whereas the red bars represent the proportion of lysogenic cells among the viable cells. The data are presented as mean ± standard deviations based on three independent experiments. NOB, Not observed.