Fig 1.
CRISPR/FnCas12a-mediated genome editing in Xoo PXO99A.
(A) Schematic illustration of pHM1B3 CRISPR cassette construction. The intermediate CRISPR construct pHZB3 is released through BamHI digestion and integrated into the vector pHM1. TetR, tetracycline resistance gene; Ptet, tetracycline-inducible promoter; FnCas12a, the CRISPR/Cas12a system from F. novicida; TrrnB, E. coli ribosomal RNA terminator rrnB; crRNA, a matured CRISPR RNA; Pj23119, a synthetic constitutive expression promoter; KanR, kanamycin resistance gene; pHM1, a broad-host-range vector; SpeR, spectinomycin resistance gene. (B) The target site of xopN. The target region in PXO99A genome is underlined and the PAM sequence is marked by the black shadow. (C) Electroporated PXO99A on agar plates. Under the same conditions, except that the control (left plate) did not contain protospacer of xopN. (D) Schematic illustration of pHM1B4 CRISPR cassette construction. It’s updated from (A) with a single operon encoding mtLigD and mtKu proteins, which reconstitute a simplified non-homologous end joining (NHEJ) machine for DSB repair in Xoo cells. (E) As for (C), but showing the genome editing system of pHM1B4. (F) Single colonies in (E) were randomly selected for preliminary identification by PCR amplification of a 1.3-kb genomic fragment flanking the xopN gene. M, 5 kb DNA ladder; Ctl, wild-type control. (G) Representative Sanger sequencing chromatogram of the deletion mutant. -77 bp, 77-bp deletion. (H) The deletion size distribution of independent mutant strains was determined by tiling PCR and Sanger sequencing. The first line is wild type, other lines represent independent mutants, the dash lines indicate the deleted region in each mutant, and the target site location is marked in red. (I) As for (H), but determined by the whole-genome sequencing (WGS) analysis of 11 PXO99A mutants. (J) The pie chart represents the proportion of different bidirectional deletion ranges. (n = 44 individual colonies randomly selected from (E)). (K) As for (J), but depicting the ratio of the different flanking micro-homology regions used for DSB repair.
Fig 2.
Multiplex gene knockout and accurate chromosomal DNA fragment deletion in Xoo PXO99A by CRISPR/FnCas12a.
(A) Schematic illustration of pHM1B4-mediated simultaneous gene editing of xopN and xopV in PXO99A with different approaches. Approach I: Equal amounts of plasmid DNA of pHM1B4-xopN-crRNA and pHM1B4-xopV-crRNA were simultaneously electroporated into PXO99A. Approach II: A single pHM1B4 plasmid carrying a crRNA array of 20- or 23-nt protospacers was electroporated into PXO99A. The relative positions of xopN and xopV genes in the PXO99A genome (NC_010717.2) are marked in red. (B) Thirty independent colonies were randomly picked for genotyping by tilling PCR amplification across ~3 kb region around the target sites. Each column represents the identities of xopN and xopV in a single independent colony. The blank, grey, and red boxes denote wild type, large deletions (>1 kb), and small deletions (<1 kb), respectively. Colonies bearing small deletions in both xopN and xopV are framed by yellow rectangles. #1–30, colony No.; crRNA-xopN & crRNA-xopV, Xoo cell transformation with two pHM1B4 plasmids (Approach I); xopN/V crRNA array, Xoo cell transformation with a single pHM1B4 plasmid carrying 20- or 23-nt protospacer (Approach II). (C) Statistical graphics showing the percentage of various deletion mutations in xopN and xopV in regard to different genome editing approaches. N, xopN; V, xopV; N&V, xopN and xopV. Colonies bearing small deletions in both xopN and xopV were considered as < 1kb category, colonies showing a mixed pattern with both < 1kb and a >1kb deletions were considered as the >1kb category, and colonies showing a mixed pattern with both wild type and >1kb deletion were considered as the WT category. No co-editing events was detected after transformation in Approach I; Co-editing of both xopN and xopV with crRNA array of 23-nt protospacers occurred in all positive colonies and resulted in large deletions. (D) Genome editing of two homologous genes (PXO_RS06285 and PXO_RS06855) using crRNA with a 20-nt protospacer. (E) Schematic illustration of precise deletion of a 3.84-kb xopAY-AV-AU gene cluster using 23-nt paired gRNAs. Surviving colonies were genotyped by PCR amplification and Sanger sequencing. The sequences below are the junction sequences with the deletions (dashed lines). The target regions are underlined and the PAM sequences are marked by the black shadow in (D) and (E). (F) Statistical graphics showing the percentage of various deletion mutations in PXO_RS06285 and PXO_RS0685585 using approach II (20-nt protospacer). A, PXO_RS06285; B, PXO_RS06855; A&B, both PXO_RS06285 and PXO_RS06855. (G) Sanger sequencing chromatograms of the deletion mutants shown in (E). The junction sequences are underlined in different colors (green and red).
Fig 3.
Iteratively knocking out all 25 non-TAL effector genes in Xoo PXO99A.
(A) A protocol for iterative rounds of genome editing in PXO99A. The pHM1B4 plasmid was transformed into PXO99A competent cells by electroporation. Surviving colonies were genotyped by Sanger sequencing of the targeted gene, and then the mutant strains that meet the requirements were selected for plasmid-curing. Determination of successful plasmid elimination was done by screening in media and PCR assay, and a plasmid-free mutant was used for the next round of knockout operations. Spe, spectinomycin; rif, Rifampicin. (B) The positions of all 25 non-TAL effectors in the PXO99A genome (NC_010717.2) are marked in red. (C) The table shows the name, target gene(s), PAM sequence, efficiency, and deletion size of the xop mutants obtained in each round of genome editing. (D) Genome-wide coverage of PXO99A when aligned back to the genome with the 212 kb duplication event (top) and the genome without the 212 kb duplication event (bottom). (E) Disease phenotypes of Kitaake after inoculation with Xoo strains. a, PXO99A; b, PXO99AΔhrcU; c, PXO99AD25E. (F) Disease lesion lengths on Kitaake. Lesions were measured 14 days post inoculation. All values represent means ± s.d. (n = 8, 5 replicates). Values with different letters significantly differ from each other (Tukey’s test, P < 0.05).
Fig 4.
CRISPR/FnCas12a-mediated genome editing in Pst DC3000.
(A) Schematic illustration of pBBR1B4-mediated gene knockout of hopAB2 residing on the circular chromosome in Pst DC3000. The relative position of hopAB2 on the chromosome is marked in red. (B) The target site of hopAB2. (C) Deletions detected in hopAB2 knockout mutants by colony PCR and Sanger sequencing. Each line represents an independent mutant, the dashed lines indicate the deleted region in each mutant, and the target site location is marked in red. (D) Schematic illustration of pBBR1B3-mediated pDC3000A plasmid curing of Pst DC3000 by targeting hopX1. The relative positions of hopX1 on pDC3000A and PCR amplification regions are shown in red and by arrows, respectively. The primer sequences are listed in the S3 Table. (E) The target site of hopX1. (F) Summary of the plasmid-curing frequency using 22 randomly-selected colonies and primer pairs shown in (D). The target regions are underlined and the PAM sequences are marked by the black shadow in (B) and (E), respectively.