Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor

Abstract

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Graphical Abstract

Figure 1.

The workflow for the design, construction, optimization and application of the dCas12a-based multiplexed dual-function base editor (MultiduBE). MultiduBE, a versatile tool for synchronized multiplexed in-situ mutagenesis, was created by amalgamating the functionalities of cytosine deaminase, adenine deaminase, and dCas12a. Through the replacement of the synthetic effector's promoter and the engineering of the crRNA array with a synthetic separator, MultiduBE demonstrates its capability to achieve simultaneous in-situ mutagenesis at five distinct genomic loci in both Escherichia coli and Bacillus subtilis. MultiduBE was employed for performing multiplexed in-situ mutagenesis, taking generic phenotypic diversity, identification of antibiotic resistance mutation, and metabolic regulation as examples.

Figure 2.

Integration of cytosine or adenine deaminases with dCas12a. (A) Schematic diagram for verifying compatibility between various cytosine deaminases and dCas12a. (B) Editing outcomes of the dCas12a-based cytosine base editors (CBEs) on target gene aprE. (C) A five-member crRNA array an5C target five sites on genes aprE and nprE was constructed to guide dCas12a-based cytosine base editors (CBEs) for multiplexed editing. (D) Editing outcomes of the dCas12a-based CBEs on target genes aprE and nprE guided by an5C. (E) Schematic diagram for verifying compatibility between evolved variants of adenine deaminase TadA and dCas12a. (F) Editing outcomes of the dCas12a-based adenine base editors (ABEs) in target genes aprE and nprE guided by an5C. Data are presented as mean values from three independent biological replicates (n = 3).

Figure 3.

Construction and optimization of the dCas12a-based MultiduBE. (A) Composition and structure optimization of the dCas12a-based MultiduBE. (B) Replacing promoter of the dCas12a-based synthetic effector in pWLBE serial plasmids generating the pWLT serial plasmids. (C) Engineering the crRNA array for improving the editing efficiency. (D) Editing outcomes on target genes aprE and nprE guided by the modified five-member crRNA arrays. (E) A crRNA array an5cN-Sp4 containing spacers of varying lengths (14- to 26-nt) was designed and assembled into pWLT-duBE-1a and pWLT-duBE-2b from multiplexed editing. Data are presented as mean values from three independent biological replicates (n = 3).

Figure 4.

Detailed editing outcomes of the MultiduBE-promoted multiplexed in-situ mutagenesis. (A) High-throughput targeted amplicon sequencing (tNGS) analysis for pWLT-duBE-1a guided by an5C or an5C-Sp4. (B) The tNGS analysis for pWLT-duBE-2b guided by an5C or an5C-Sp4. The top 10 alleles produced from an5C and an5C-Sp4 in each target were selected and compared. The asterisk means C > T or A > G conversion occurred in the complementary DNA strand of protospacer. The alleles marked in blue mean the bystander editing out-of-protospacer (BEOP), and those with red underline mean combinatorial C > T and A > G mutants. The short lines represent mean values from three independent biological replicates (n = 3), and the circles represent individual data points. More than 500 000 reads were used for each sample. See Supplementary Table S3 for details including the reads of NGS.

Figure 5.

Multiplexed in-situ mutagenesis for cell physiology reprogramming. (A) To validate MultiduBE in generic phenotypic diversification, strain G00-CmYKT was constructed by integrating three fluorescent proteins (sYFP2, mKate, and mtagBFP2) into the genome of strain G00. For cellular morphology reprogramming, FtsZ (tubulin to form Z-ring at the midcell) and MreB (cytoskeleton protein) were selected as the targets for MultiduBE. (B) The target table containing designed crRNAs for verifying MultiduBE in generic phenotypic diversification. (C) The five-member crRNA array YKTmc5C-Sp4 guided MultiduBE in generic phenotypic diversification. After treatment by MultiduBE, confocal images of strain G00-CmYKT were obtained by merging the brightfield, green (500–550 nm), red (570–616 nm) and blue (429–474 nm) channels. (D) The target table containing designed crRNAs for antibiotic (tetracycline, rifampicin, spectinomycin, or streptomycin) resistant mutant generation. Nucleotides marked in red indicate potential mutation sites based on previous studies. (E) Sequencing analysis of the crRNA arrays and the genomic targets. (F) Based on the crRNA arrays enriched during the screening process, a four-member crRNA array LBEL4C-Sp4 was designed to confer resistance to the four types of antibiotics used. M1–M3 represent the corresponding mutations for each gene, and details can be found in Supplementary Figure S16C. (G) Approximately 10⁸ cells were diluted with distilled water, and 5 μl aliquots of the cell suspension were spotted onto the agar plate with specific antibiotics. (H) Growth curves of the wild and mutated strains. Data are presented as mean values ± SD from three independent biological replicates (n = 3). Lines indicate the mean, and shaded areas represent the SD.

Figure 6.

Multiplexed in-situ mutagenesis for metabolic regulation. (A) The target table containing designed crRNAs for surfactin synthesis regulation. (B) A five-member crRNA array sur5C-Sp4 was constructed and utilized for multiplexed in-situ mutagenesis on genes related to surfactin synthesis. Using a colorimetric method, 12 mutated strains (Sur-s1∼s12) were screened. (C) Strains G600, Sur-s0, Sur-s1, Sur-s11 and Sur-s12 were selected for shake-flask production of surfactin. M1 represents the corresponding mutation for each gene, and details can be found in Supplementary Table S4. Data are presented as mean values ± SD from three independent biological replicates (n = 3). The circles represent individual data points of surfactin titer, and the squares represent individual data points of OD₆₀₀. The chromatogram of the surfactin standard (including four surfactin isoforms) is shown in yellow with a light shadow, while the surfactin chromatograms of selected strains are shown in the colors corresponding to the circles. (D) The synthetic pathway and regulation manner of riboflavin in B. subtilis. (E) The target table containing designed crRNAs for the emergence of the riboflavin overproducer. Nucleotides marked in red indicate potential mutation sites based on previous studies. (F) A five-member crRNA array rib5C-Sp4 was constructed to facilitate multiplexed in-situ mutagenesis for the emergence of riboflavin overproducer from strain G600. Fluorescence-activated droplet sorting (FADS) was conducted to screen the mutations overproducing riboflavin. (G) The isolated single colonies were picked and screened by fluorescence measurement in 96-well plates. (H) Five mutated strains (Rib-s4, Rib-s8, Rib-s11, Rib-s12 and Rib-s14) were selected for shake-flask culture. The strain Rib-s0, constructed by replacing P_ribD and FMN riboswitch with a strong constitutive promoter P_veg, was used as a control. M1–M5 represent different mutations for each gene, and details can be found in Supplementary Table S5. Data are presented as mean values ± SD from three independent biological replicates (n = 3). Lines indicate the mean and shaded areas represent the SD.