Junctions 1 and 2 (j1 and j2) are new junctions. (iii) Lambda-red recombination joins fragments and linkers to yield chromosomes 1 and 2 (Chr. (ii) Homology regions (HRs) between fragments and their cognate linkers. 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). coli harbors a fission BAC, containing a double selection cassette ( sacB-Cm R shown as pink and green, respectively), rpsL (yellow), a luxABCDE operon (white), and the BAC replication machinery (orange). S1) this linker sequence contained its own replication and segregation machinery. Similarly, chromosome 2 (0.56 Mb) was formed through lambda-red-mediated recombination between genomic fragment 2 and linker sequence 2 ( Fig. Chromosome 1 (3.43 Mb) containing the genomic origin of replication ( oriC) was formed through lambda-red-mediated recombination between genomic fragment 1 and linker sequence 1, by virtue of their 50-base pair (bp) regions of homology ( table S2).
The two cuts in the genome create fragment 1 and fragment 2, and the four cuts in the fission BAC release linker sequence 1 and linker sequence 2. We implemented six Cas9-directed cuts in the DNA of these cells two of these cuts target the genome, and four of these cuts target the fission BAC ( data files S3 and S4). To achieve this, we first introduced Cas9 with appropriate spacers ( table S1), the lambda-red recombination machinery, and a fission bacterial artificial chromosome (BAC) ( data file S2) into cells. coli MDS42 ( 1) genome ( data file S1) into a 3.43- and a 0.56-Mb chromosome. 1A) and tested our approach by splitting the E. We designed and synthesized a system to precisely split the unmodified genome into two user-defined, circular chromosomes ( Fig. The resulting synthetic chromosomes enable precise, programmed fusions, genomic inversions and translocations moreover, they provide a route to assemble new genomes through the precise, convergent assembly of large genomic fragments from distinct strains.
coli genome, without any prior modification, can be efficiently split, by single-step programmed fission, into pairs of synthetic chromosomes. coli genome are not general or efficient. Thus, the limited methods for splitting the E. However, only one out of 22 arrangements tested was viable ( 21). coli chromosome into two linear subchromosomes. A protelomerase of bacteriophage N15 and a Vibrio origin of replication were used to divide the circular E. coli is generally low ( 20), this approach is presumably very inefficient. Because the recombination frequency in E. coli genome ( 19) by using natural homologous recombination in E. One report excised up to 720 kb from a single region of the E. The synthetic splitting and fusion of chromosomes has been explored to a limited extent, primarily in naturally recombinogenic organisms ( 13– 18). Indeed, in favorable cases, crossovers are only selected with kilobase resolution.Ĭhromosome fission and fusion have occurred in natural evolution ( 10, 11), and these processes may have accelerated evolution ( 10, 12, 13). While these methods can be useful ( 5, 9), they are fundamentally limited because (i) they require large regions of homology, (ii) undesired chimeras between the two genomes may result, and (iii) the site of crossover between the two genomes is not precisely specified. Moreover, methods for combining large (e.g., 0.5-Mb) sections from distinct genomes rely on classical conjugation ( 8) and its derivatives ( 5, 9). coli, with most current methods for inversions relying on sequence-specific recombinases. Each operation should be scarless and programmed with nucleotide precision so that genome designs can be precisely and rapidly realized.Įfficient, precise, and robust methods for iterative replacement (>100 kb per step) and deletion of genome sections have been reported ( 7) however, there has been less progress on creating methods for generating precisely programmed inversions or translocations in E.
These operations include (i) the iterative replacement of genomic DNA with synthetic DNA, (ii) deletion of genomic DNA, (iii) translocation of large genomic sections, and (iv) inversion of large genomic sections as well as (v) methods for combining large genome sections from distinct strains for the convergent assembly of synthetic genomes. However, in Escherichia coli, the workhorse of synthetic biology, the methods necessary to realize a complete set of operations for synthetic genome design are missing. Efforts to minimize ( 1, 2), refactor ( 3), recode ( 4, 5), and reorganize chromosomes and genomes ( 2, 6) are providing new insights and opportunities.
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