Fig. 1 Programmed genome fission splits the E. coli genome into two chromosomes. (A) E. coli harbors a fission BAC containing a double selection cassette (sacB-CmR shown as pink and green, respectively), rpsL (yellow), a luxABCDE operon (white), and the BAC replication machinery (orange). During fission, (i) Cas9 induces six cuts (black triangles), splitting the genome into fragment 1 (light gray, containing oriC indicated by black line) and fragment 2 (dark gray) and the fission BAC into four pieces (linker sequence 1, linker sequence 2, and two copies of rpsL). (ii) Homology regions (HRs) between fragments and their cognate linkers. (iii) Lambda red recombination joins fragments and linkers to yield chromosomes 1 and 2 (Chr. 1 and Chr. 2). Junctions 1 and 2 (j1 and j2) are new junctions. (B) Growth and luminescence (Lumi.) of prefission (pre) and postfission (1 and 2) clones are consistent with the generation of two chromosomes (Chr. 1, ~3.43 Mb and Chr. 2, ~0.56 Mb). Cells were stamped in plain LB agar (-), 20 μg/ml chloramphenicol (Cm), 7.5% sucrose (Suc), 100 μg/ml streptomycin (Strep), or the indicated combination. (C) PCR of postfission (Post-Fiss.) clones across j1 and j2.
Fig. 2 Fission can be performed throughout the E. coli genome. (A) Successful fissions performed. Each color on the E. coli genome corresponds to ~0.5 Mb. We named the sections A to H. A is dark orange, and the other sections are labeled alphabetically in a clockwise sequence. Linker sequence 1, white; oriC, black bar; linker sequence 2, gray. Boundaries and homologies of each fission experiment are provided in table S2. Seven fissions are shown, including the 3.43, 0.56 Mb fission (Fig. 1). The 3.45, 0.54 Mb fission (purple Chr. 2) was performed by using an E .coli genome in which a ~0.54-Mb section had been recoded (Fig. 3). (B) Growth and luminescence for the generation of the 2.44, 1.55 Mb fission; annotation as in Fig. 1B. Data for other fissions are shown in fig. S4. (C) PCR of clones across new junctions for 2.44, 1.55 Mb fission. Postfission clones (1 to 5) exhibit products of the expected size, whereas the prefission control does not. Junction PCRs for other fissions are in fig. S4.
Fig. 3 Programmed chromosomal fusion enables translocations and inversions of large genomic segments from common fission intermediates. (A) E. coli with two chromosomes (Chr. 1 ~3.45 Mb and Chr. 2 ~0.54 Mb) was generated by fission. The sequence of Chr. 2 is watermarked as described in the text. The color-coding is as in Fig. 1A; a pheS*-KanR double selection cassette (purple and yellow, respectively) is shown. A fusion sequence, consisting of a pheS*-HygR (purple and blue, respectively) double selection cassette flanked by HR1 and HR2, is introduced in the indicated positions and orientation in Chr. 1 by lambda-red recombination. Cas9 spacer-directed cleavage (black arrows), lambda-red recombination, and selection for fusion products through the loss of pheS* on 4-chloro-phenylalanine yield the indicated products. (i) Regenerating the original genomic arrangement, (ii) translocation of the 0.54-Mb segment 700 kb away from its original position, and (iii) inversion of the 0.54-Mb segment. (B) Growth and luminescence of pre- and postfusion regeneration (1 and 2) clones. Hyg, hygromycin; Kan, kanamycin; p-Cl-Phe, 4-chloro-phenylalanine. (C) PCR of clones across new junctions for fusion regeneration. Postfusion clones (1 to 8) exhibit products of the expected size, whereas the pre-fusion control does not. wt, wild type. (D and E) As in (B and C) but for fusion translocation (trans.). (F and G) As in (B and C) but for fusion inversion (inv.).
Fig. 4 Precise genome assembly from genomic segments of distinct strains. (A) Precisely combining the watermarked region 1 (dark gray) from a donor strain and a watermarked region 2 (black striped) from a recipient strain into a single strain. Fission is performed in parallel in the donor and recipient strains. The resulting donor strain contains a watermarked Chr. 2 containing an oriT (black arrow) and a pheS*-KanR double selection cassette (purple and yellow); the remainder of linker sequence 2 is orange. The resulting recipient strain contains an analogous nonwatermarked Chr. 2, with a sacB-CmR cassette (pink and green). The linker sequence 1 (white) is replaced with a fusion sequence containing a pheS*-HygR cassette (purple and blue) in preparation for fusion. The donor cell is provided with a nontransferable F′ plasmid. Mixing of donor and recipient cells facilitates conjugative transplant of Chr. 2 from the donor to the recipient; selection for KanR and against sacB-mediated sucrose sensitivity enables the isolation of cells that have gained a watermarked Chr. 2 and lost the nonwatermarked Chr. 2. Subsequent genome fusion generates a strain in which the watermarked regions 1 and 2 have been precisely combined in a single chromosome. (B) Following the process of chromosomal transplant by growth on selective media and luminescence. d, the pretransplant donor; r, pretransplant recipient. (C) Following the process of chromosomal fusion through growth on selective media. (D) PCR across the new junctions generated by chromosomal fusion yields products of the expected size in the postfusion clones (1 to 10) but not in the prefusion control.
Supplementary Materials
science.sciencemag.org/content/365/6456/922/suppl/DC1
Materials and Methods
Figs. S1 to S8
Tables S1 to S6
Data Files S1 to S7
References (23–27)
Additional Files
- Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly
Kaihang Wang, Daniel de la Torre, Wesley E. Robertson, Jason W. Chin
Materials/Methods, Supplementary Text, Tables, Figures, and/or References
- Download Supplement
- Materials and Methods
- Figs. S1 to S8
- Tables S3 to S5
- References
- Table S1
- Table S2
- Table S6
- Data Files S1 to S7
