A multifunctional system for genome editing and large-scale interspecies gene transfer

CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. We demonstrate the excision, transfer and integration of large chromosomal regions between E. coli, V. natriegens and D. dadantii without size-limiting intermediate DNA extraction. CRISPR SWAPnDROP also provides common genome editing approaches comprising scarless, marker-free, iterative and parallel insertions and deletions. The modular character facilitates DNA library applications, and recycling of standardized parts. Its multi-color scarless co-selection system significantly improves editing efficiency and provides visual quality controls throughout the assembly and editing process.


Introduction
In the recent years, the relevance of genome editing in bacteria rapidly increased in basic research, biotechnology and synthetic biology.Novel technologies like CRISPR/Cas9 and large scale DNA synthesis brought genome editing within reach of a broad scientific community.However, large-size modifications, incompatibility of individual tools as well as high-throughput systems are still challenging.The basic steps of genome editing in bacteria is the introduction of exogenous template DNA in the form of plasmids, ssDNA or dsDNA and the subsequent integration into the chromosome via homologous recombination.Homologous recombination is a very inefficient process, even when improved with recombination systems like λ RED.Different strategies have been developed to overcome time consuming screening for edited cells, e.g. the integration of antibiotic resistance markers or metabolic markers [1][2][3][4] .However, the number of possible edits in one cell is limited by the number of available markers as only one edit per marker is possible.Additionally, the introduction of additional marker genes into the chromosome can cause undesired interference with adjacent transcription units 5,6 .To circumvent the problem of marker limitation, site-specific recombinases such as Cre-and FLP recombinase are introduced to remove the selection marker after chromosomal integration 7,8 .This adds an additional step to the editing process and leaves active recombination sites in the genome, which eventually limits the application of this approach even with the use of alternative recombination sites 9 .Another strategy to avoid selection markers and other scars is oligo-mediated allelic replacement (OMAR).Short single-stranded (ss) DNA are incorporated into the genome by an Okazaki-like allelic-replacement event at the replication fork and facilitate point mutations, small deletions and insertions 10 .Automation and cyclizing of this method allows genome modifications of several genes 11 .However, OMAR is limited by the size of ssDNA oligos used and therefore larger edits are not possible.For larger scarless genome editing, counter-selection methods can be applied.Such methods rely on efficient counter-selectors like the meganucleases I-SceI and I-CreI, which introduce double-strand breaks at a specific site of 18bp and 22bp in length 12,13 .The double-strand break leads to cell death of non-edited cells.This strongly enriches the viable population for successfully edited cells.However, the approach requires an additional classical editing step, in which the meganucleases' target site is first integrated into the chromosome at the site of interest.The discovery of CRISPR/Cas9 abolished the initial integration of a specific restriction site.Similar to a meganuclease, Cas9 is an endonuclease.In contrast to a meganuclease, Cas9 has no DNA binding specificity.DNA

Sequencing of Bacterial Genomes and Data Analysis
Isolation of bacterial genomic DNA was performed according to Bruhn et al. 26 .Next-generation sequencing (NGS) was done by Eurofins Genomics Germany GmbH (Ebersberg, Germany).Reads were mapped and coverage was determined by the R package QuasR using a custom R script.The mutant reference genomes used for mapping were made using SnapGene on the basis of the E. coli MG1655 genome.

pSwap Assembly
The pSwap plasmid consists of seven modules each harbouring a part for the Swap and Drop recombination system: T1, T2 (sgRNA expression cassettes); H1α, H2β , HA, HB (homologous regions) and INS (insert fragment).For the T1 and T2 construction, 1µl of complementary oligonucleotides (10µM) consisting of the 20bp target sequence and corresponding overhangs (see Table 1), 10µl of 10X T4-Ligase buffer (NEB) and 88µl MilliQ water were incubated at 95°C for 10 minutes and then slowly cooled down to room temperature.1µl of annealed oligonucleotides were used for cloning.For the construction of the other parts, PCR products using primer with corresponding overhangs were used (see Table1).Approximately 40fmol (∼25ng/1000bp) of PCR products were utilized for cloning.Annealed oligonucleotides or PCR products were mixed with 40fmol of the corresponding entry vectors, 1X T4-Ligase buffer (NEB), 1µl (20 units) BsaI-HFv2 (NEB), 1µl (400 units) T4-Ligase (NEB) and MilliQ water to a final volume of 20µl.The mix was incubated for 2-5 hours at 37°C for in vitro assembly.In case of internal BsaI sites in the inserts, a final 30 minutes ligation step at 16°C prior to heat inactivation is added for optimal

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Ligase activity and reduced BsaI activity.The mix was transformed into chemically competent DH5α cells and subsequently plated on LB Agar supplemented with 1mM IPTG and 250µM X-gal.White colonies were tested for a successful integration into the entry vectors via sanger sequencing.Antibiotic concentrations used for cultivation of each part is seen in Table1.If needed, Hα-and Hβ homologies for H1α and H2β were added as an additional PCR fragment without fixed overhangs upstream of the 5'-end of the H1 homology or downstream of the 3'-end of the H2 homology.For the pSwap assembly, 40fmol of each part (T1, T2, H1α, H2β , HA, HB, INS), 1X T4-Ligase buffer, 1µl (10 units) BbsI (NEB), 1µl (400 units) T4-Ligase and MilliQ water were incubated in a final volume of 20µl for 5-7 hours at 37°C.In case of internal BbsI sites in the inserts, a final 30 minutes ligation step at 16°C prior to heat inactivation can be added for optimal Ligase activity and reduced BbsI activity.The restriction-ligation could then be transformed into a cloning (DH5α) or directly into the desired wild-type strain.Succesfully assembled pSwap plasmids were verified by purple colonies after transformation.Different parts of the deoxyviolacein pathway are distributed on different modules.However, only when the modules are correctly assembled into the pSwap, a functional deoxyviolacein pathway result in purple colonies.Antibiotics were used depending on the corresponding H2β (see Table1).For insertion or deletion experiments, T2 was not essential, but still mandatory for the pSwap assembly.Therefore, a random DNA sequence, which was not present in the strains' chromosomal sequence, was cloned into the entry vector and used for the assembly.Homologies (H1, H2) for the loading of the pSwap were also not needed, but a recombination between those random sequences was important to maintain the integrity of the pSwap plasmid during genome modifications.Therefore, identical 175bp random DNA sequences were cloned into H1α and H2β (HR, see Fig. 1A).In general homology arms of 500bp 27 and 1500bp 28 were used for E. coli and V. natriegens, respectively.As all parts were digested with BsaI and BbsI for cloning, the presence of those restriction sites within the parts (e.g. the homologies or insert fragment) were avoided.Therefore, in some cases the size for homology arms were chosen between 50-500bp for E. coli and between 1000-1500bp for V. natriegens to avoid present restriction sites.A list with Primers, which were used for the construction of each individual pSwap plasmid can be found in the supplementary data.CRISPR SWAPnDROP Genome Editing Protocol (Indel/Swap) E. coli MG1655 was first transformed with the helper plasmid cr3Ec.For all CRISPR/Cas9 experiments, cr3Ec harbouring the P tet repressor (TetR) needed to be transformed first to establish P tet repression prior to the transformation with pSwap.Double transformation with pSwap and cr3Ec lead to disfunctional components in the Cas9 system 14 .After transformation with cr3Ec, E. coli MG1655 cr3Ec was transformed either directly with the pSwap restriction-ligation mix or the prepared pSwap plasmid.The transformants were grown on LB Agar with appropriate antibiotics (see Table2) at 37°C over night.The plates were then incubated for 2-5 hours at room temperature allowing the colonies to form a purple color.Subsequently, purple colonies were inoculated in LB with 100 ng/ml Anhydrotetracyclin (aTet), 1% arabinose and appropriate antibiotics (see Table2).Cultures were grown at 37°C shaking (200rpm) over night and plated on LB Agar with the same antibiotics at 37°C.After approximately 16 hours, the plates were incubated at room temperature for 2-5 hours now allowing the colonies to form a green color.Green colonies were then tested for a successful editing event via PCR and Sanger sequencing.

Vibrio natriegens Genome Editing Protocol (Indel/Swap)
The V. natriegens strain ATCC 14048 ∆VNP12 was first transformed with cr3Vn.Subsequently, this strain was transformed with pSwap Cm R and incubated on LBv2 Agar with corresponding antibiotics at 37°C over night (see Table3).The plates were 3/26 incubated at room temperature for several hours to develop its purple color.Purple colonies were inoculated and grown until OD 600 reached 4 or over night in LBv2 with appropriate antibiotics at 37°C and 200rpm shaking.Induction of the λ RED genes was carried out by adding 0.4% arabinose for 1-2 hours followed by the induction of the CRISPR/Cas9 genes with aTet (80ng/ml) for further 2-4 hours at 37°C.Subsequently, the cultures were plated on LBv2 with 80ng/ml aTet, 0.4% arabinose, antibiotics (see Table3) and grown at 37°C over night.The following day, colonies were incubated at room temperature for several hours or until the next day.Green colonies were then tested via PCR and Sanger sequencing.

Conjugation and Drop Protocol
For the transfer and integration of chromosomal DNA, the loading of the pSwap plasmid with a chromosomal region was carried out in the donor strain E. coli MFDpir ∆dapA ∆endA , harbouring the RP4 conjugation system 23 .Simultaneously, the acceptor strain was transformed with the helper plasmids cr3Ec and pDrop.Similar to the sgRNA level 1 parts (T1, T2) of the pSwap assembly, the spacer sequence (T3) for the counter-selection in the acceptor strain needed to be cloned into the pDrop entry vector and subsequently verified by sanger sequencing.Both, the donor strain harbouring the loaded pSwap plasmid (pSwap') and the RP4 conjugation system as well as the acceptor strain harbouring the helper plasmids were cultivated in LB with correct antibiotics at 37°C over night (see Table2).The culture of the donor strain was additionally supplemented with 300µM diaminopimelic acid (DAP).The donor and acceptor strains were diluted to an OD 600 of 3 with fresh LB medium after washing and then mixed with a 1:1 ratio.Subsequently, the mix was spotted on LB Agar plates only supplemented with 300µM DAP and incubated at 37°C over night.The spot was scrapped off the plate and washed several times with LB medium to remove residual DAP.Selection for successful conjugation was performed on LB Agar with appropriate antibiotics (see Table2).After the conjugation of the pSwap' plasmid, the acceptor strain was inoculated and directly induced in LB with the corresponding antibiotics for the selection of cr3Ec and pDrop (see Table2).Selection for the pSwap was omitted, as during the excision of the recombination template, the pSwap plasmid was destroyed.Correct function of the CRISPR/Cas9-and λ RED-systems was ensured by the cut and reconstitution of the pDrop non-functional mScarlet gene yielding red colonies.Induction was again carried out with 1% arabinose and 100 ng/ml aTet.The induced acceptor strain was cultivated over night at 37°C and then plated on LB with the same antibiotics.Red colonies were tested for the integration into chromosome via PCR and sanger sequencing.Conjugation and subsequent chromosome integration from E. coli to V. natriegens was carried out similarly.For V. natriegens pDropVn containing a gentamycin resistance cassette was used, as selection with ampicillin did not provide satisfying results.For the conjugation, the stationary phase cultures diluted to an OD 600 of 3 were mixed with a 1:9 ratio (V.natriegens : E. coli) and then spotted on LBv2 agar plates, also supplemented with DAP.V. natriegens now harbouring the pSwap' plasmid, cr3Vn and pDropVn were inoculated and grown over night at 37°C with corresponding antibiotics (see Table3).The next day, induction was performed in the stationary phase culture by inducing first the λ RED recombination with 0,4% arabinose for 1-2 hours and then the CRISPR/Cas9 with 80 ng/ml aTet for another 2-4 hours.The induced culture was then plated on LBv2 agar with antibiotics as well as 0.4% arabinose and 80 ng/ml aTet.Red colonies were tested via PCR and Sanger sequencing.

Results
DNA synthesis technologies and modular cloning approaches allow for the assembly of large DNA fragments at a scale of bacterial chromosomes.Such large fragments play a role in biotechnology for complex pathway assembly or tailored organism design.Moreover, in synthetic biology and basic chromosome research, rearranged or even completely synthetic chromosomes receive increasing attention 16,29,30 .Such approaches, however, require reliable and versatile handling of large DNA fragments and DNA libraries.We have developed CRISPR SWAPnDROP to meet these requirements.CRISPR SWAPnDROP is based on homologous recombination, CRISPR/Cas9 counter-selection and a scarless multi-color co-selection system.CRISPR SWAPnDROP provides a framework for the assembly of large genomic sequences, the rearrangement of chromosomal parts as well as the sequence transfer between strains and even to other organisms (see Fig. 1).Furthermore, it comes with a full set of editing tools for convenient iterative and parallel scarless chromosomal deletions and insertions.Table 4 provides an overview of the whole workflow regarding days, tasks and expenditure of time.

Concept
CRISPR SWAPnDROP is based on homologous recombination of linear double stranded DNA.It allows for insertion and deletions as well as transfer of large DNA fragments between strains or species (Fig. 1).For insertions and deletions (Indel) two flanking homologies (HA, HB) are used for chromosomal integration of the insert (INS).The fragment HA-INS-HB is released from a plasmid inside the cell by restriction with Cas9.Furthermore, Cas9 is used as a counter-selector using locus-specific sgRNAs expressed from the same plasmid.For the transfer of DNA between cells, chromosomal DNA is loaded onto the plasmid by another set of homologies (H1, H2) matching the flanks of the chromosomal fragment (Swap).Fragment excision and plasmid opening is mediated by Cas9 and sgRNAs specific for the flanks of the chromosomal fragment and the plasmid.
After loading the fragment onto the plasmid, it is transferred to another cell via RP4 conjugation or plasmid transformation.
In the new host, in analogy to the insertion process, the fragment is released by flanking Cas9 restriction and another pair of homologies (Hα, Hβ ) located at the edges of the fragment confer recombination (Drop).The editing process is supported by a color system based on the deoxyviolacein pathway and the mScarlet gene that provides positive feedback of the experimental state in each step of editing by changing colors (Fig. 1A).Cells harbouring the correct assembled pSwap plasmid produce a purple color.Cells, which successfully performed Cas9 restriction and homologous recombination during the Indel/Swap and the Drop steps produce a green and red color, respectively.For CRISPR SWAPnDROP to function properly it is central that recombination and Cas9 restriction works efficiently.Although the CRISPR SWAPnDROP concept described above is generally applicable, it is apparently not possible to provide a static system that will be functional in many species.Given the diversity of species, central components like origins of replication, resistance cassettes, recombination systems or individual promoters and RBS need to be adapted to the individual species.Therefore, we have designed CRISPR SWAPnDROP in a highly modular fashion to allow for efficient combinatorial screenings for the adaptation to new species.In this study, we show the adaptation process for the model organism E. coli and the fast growing marine bacterium V. natriegens.Details about the features, mechanisms, helper plasmids and modularity are provided in the following paragraphs.

cr3 -The CRISPR SWAPnDROP Workhorse
The cr3 helper plasmid harbours the enzymes required for genome editing.These enzymes comprise the Cas9 endonuclease and a species-specific recombination system.The plasmid itself is assembled via the MoCloFlex system 20 .With the MoCloFlex system, the assembly of up to 5 components in any orientation, order and composition is possible in one step.This allows the user to adapt the cr3 plasmid to different species if necessary by exchanging parts e.g origin of replication, antibiotic resistance or the recombination system.In this study, we show the composition of cr3 for E. coli (cr3Ec) and V. natriegens (cr3Vn).

pSwap -The Modular Carrier Plasmid
The pSwap plasmid represents the variable component of the CRISPR SWAPnDROP system.It harbours all edit-specific parts including insertion fragments, sgRNAs and homologous regions for the integration and transfer of DNA.The assembly is based on modular cloning and confers edit-specific customization of the pSwap plasmid.

pDrop -The Chromosomal Transfer Helper Plasmid
The pDrop plasmid supports the integration of the transferred chromosomal DNA into the new chromosomal locus.It consists of a fixed and a flexible sgRNA, which are responsible for the in-vivo excision of the transferred DNA for homologous recombination and the counter-selection for a successful integration, respectively.

Cas9-based Selection and Recombination
CRISPR SWAPnDROP can be used for chromosomal modifications such as insertions and deletions (Indels) as well as for the rearrangement and transfer of chromosomal regions between bacterial strains (see Fig. 1).For the creation of Indels,

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the template for homologous recombination is located on the pSwap plasmid consisting of homologous regions HA and HB flanking the desired insert (INS).Induction of the CRISPR/Cas9 and the recombination system leads to the excision of the template and its integration into the chromosome.Recombination of the homologous regions HR, which flank the excised fragment (HA-INS-HB) and have the same nucleotide sequence, recircularize the pSwap plasmid after excision of the template DNA.This is required to maintain plasmid integrity when no loading of the pSwap for DNA transfer is required (see Fig. 1A).Expression of the T1 sgRNA/Cas9 targeting the chromosomal region allows selection for a successful recombination event.Only cells which lost the target site upon integration of the excised fragment survive.For scarless deletions, it is possible to omit the INS fragment leaving only the homologous regions HA and HB.For the transfer of chromosomal regions, the pSwap can also be loaded with chromosomal DNA.In this case, the regions H1 and H2 are homologous to the flanking regions of the chromosomal region to be transferred (see Fig. 1B).Expression of T1 and T2 sgRNA/Cas9 as well as TS sgRNA/Cas9 cause excision of the chromosomal region and the HA-INS-HB fragment, respectively.The recombination system then catalyzes the swap of both DNA fragments leading to a loaded pSwap (pSwap') with chromosomal DNA and a chromosome with a deletion or a substitute fragment.Here, the sgRNAs act as excision and selection tools both on the chromosome and the pSwap plasmid.For dropping the loaded sequence at the desired location, the swap approach can be further extended by an additional set of homologies Hα and Hβ that flank the region of integration.This is supported by the helper plasmid pDrop expressing sgRNAs TD and T3, which are used for the in-vivo excision of the loaded DNA and the selection at the insertion locus, respectively (see Fig. 1C).

Modular Assembly of the pSwap Plasmid
The pSwap plasmid consists of seven locus-specific parts necessary for genome editing, e.g.homologous regions (HA, HB, H1, H2, Hα, Hβ ), sgRNA expression cassettes for counter-selection and excision (T1, T2) or insert DNA (INS).Each part has its own vector that can be joined into a pSwap plasmid with the desired parts, allowing for the recycling of parts, e.g. the recycling of homologies and sgRNAs to insert different sequence at the same location, or the recycling of the insert at different locations (see Fig. 2A and B).This approach reduces cloning efforts and supports the storage-efficient implementation of a parts library.Moreover, for the pSwap plasmid, assembled from sequenced parts, no additional sequencing is required.The pSwap assembly follows a simple hierarchical topology similar to other cloning systems 19,20 and is also based on Golden-Gate cloning.Each of the seven modules has its specific level 1 entry vector for cloning the desired parts (see Fig. 2A).Additionally, the INS entry vector is level 1 MoClo 19 , MoCloFlex 20 and Marburg Collection 22 compatible.Hence, level 1 transcription units build with the MoClo-System or Marburg Collection as well as larger assemblies build with MoCloFlex can be cloned into the INS entry vector.Therefore, already present libraries for these systems can be accessed by CRISPR SWAPnDROP and used for chromosomal integration.Except for the H2, each entry vector contains a high-copy pUC origin of replication, a kanamycin resistance cassette and all contain the ccdB and lacZα genes for selective cloning 31 .The selective cassette is flanked by two BsaI restriction sites for Golden Gate cloning of the desired DNA fragments.As BsaI is a type-IIS restriction enzyme, cleavage occurs outside of its recognition sequence and therefore corresponding sites are lost upon restriction.This allows cloning in a one-pot, one-step reaction, in which the selective cassette is removed and replaced with the desired DNA fragment.Each of the resulting level 1 vectors contain two additional type-IIS BbsI restriction sites flanking the cloned DNA fragment for the assembly of a level 2 pSwap plasmid.All necessary level 1 modules are cleaved with BbsI and the DNA fragments are assembled via DNA ligase in a one-pot, one-step reaction due to fixed overhangs (see Fig. 2B).In addition to the other level 1 entry vectors, the different versions of the H2 entry vector (Cm R , Gm R ) contain a single copy F-origin of replication and a chloramphenicol or gentamycin cassette for the final pSwap.The single-copy H2 plasmid also contains a RP4 origin of transfer for conjugation of large genomic regions.For the scarless integration into the chromosome, HAIB plasmid can be used replacing the modules HA, INS and HB.A standard assembly of pSwap comprises 7 parts.With a growing number of parts in a golden gate reaction, the number of correctly assembled plasmids decrease 20 .The selection for a correctly assembled pSwap is guided by co-expression of the deoxyviolacein pathway, whose individual genes are distributed among the parts T1, T2, H1α and HA.A successful assembly results in the formation of purple colonies after transformation (see Fig. 2C,E and Fig. S1A).This allows the direct transformation of wild-type strains without an additional assembly step using a cloning strain.Within this study, all selected purple colonies lead to successful edits.A detailed plasmid map of pSwap is depicted in Figure 3.

Design and Construction of the cr3Ec and cr3Vn Plasmids
The construction of the cr3Ec and cr3Vn plasmids were carried out using the modular cloning systems MoCloFlex 20 and the Marburg Collection 22 .The Marburg collection was used to assemble individual transcription units from promoter, RBS, CDS, tag and terminator parts in the library.Additionally, the library was extended with newly designed parts.From the Marburg collection, the origin of replication as well as resistance marker, cas9 and recombination transcription units were transferred into position vectors AB, CD, EF and IJ of the MoCloFlex (MCF) system, respectively.The position vectors together with MCF linkers BC, DE, FI and JA allowed us to assemble a set of cr3 variants, each harbouring different combinations of origins of replication, resistance cassettes and induction systems for CRISPR/Cas9 and homologous recombination.The variants were 6/26 then screened for compatibility with the targeted organism and the other helper plasmids pSwap and pDrop.For the construction of the cr3Ec, the broad host range origin of replication RSF1010 (MCF RSF1010 origin of replication), a kanamycin resistant cassette (MCF KanR), Cas9 transcription unit (MCF Cas9) and a λ RED transcription unit (MCF RED) (see Fig. 3) was assembled.In accordance with Reisch and Prather 14 , the Cas9 transcription unit was assembled using an inducible tetracycline promoter (P tet ), a weak ribosomal binding site, which was integrated as a level 0 part into the Marburg Collection library, the Cas9 coding sequence, the M0051 ssrA degradation tag and the B0015 terminator.For the Cas9 coding sequence, Esp3I, BbsI and BsaI recognition sites were removed by introducing silent mutations to confer compatibility with the Marburg Collection, MoCloFlex and CRISPR SWAPnDROP Golden Gate systems.For efficient homologous recombination, the λ RED system was used.Its transcription unit was assembled using the promoter part harbouring the P BAD and araC of E. coli, the Gam, Beta, Exo coding sequence and and the B0015 terminator 22 .For the construction of the cr3Vn, the broad host range RSF1010 as well as a codon-optimized kanamycin resistance gene were combined, which were known to ensure plasmid stability in V. natriegens 22 .In earlier studies, phage-derived or native recombination systems were used to confer efficient homologous recombination in V. natriegens 28,32 .In this study, we modified the E. coli λ RED operon by setting it under the control of the native V. natriegens P BAD promoter to support homologous recombination.The repression was conferred by the native araC gene located on the chromosome.

Enhanced Editing Efficiency by Multi-color Scarless Co-selection
CRISPR SWAPnDROP enhances editing efficiency by a multi-color scarless co-selection strategy.This co-selection strategy selects for functional CRISPR/Cas9 and recombination systems, thus decreasing suppressor mutations (see Table 5).Consequently, editing efficiency is increased without scars or the trade off of additional chromosomal markers 33 .The CRISPR SWAPnDROP co-selection is based on the elimination of the vioC gene from the pSwap plasmid by CRISPR/Cas9 mediated excision and subsequent recombination upon induction (see Fig. 2D).The lack of vioC leads to a metabolic shift towards pro-deoxyviolacein and, in turn, to the formation of green colonies (see Fig. 2E).To assess the gene editing efficiency of CRISPR SWAPnDROP two E. coli genes, lacZ and araB, were reconstituted and phenotypically tested.For that, two E. coli knockout strains were first generated each lacking either a functional lacZ or araB gene by deleting parts of the corresponding coding sequence and introducing Cas9 target sites.Deleting only parts of the coding sequence renders the genes non-functional.At the same time it ensures that the reconstituting fragment on its own does not restore gene function in the subsequent reconstitution step.In this way, neither the pSwap plasmid containing the reconstituting fragment nor an off-target insertion into another locus causes false positive results.The genes lacZ and araB encode for the β -galactosidase and ribulokinase, both essential for the lactose and arabinose metabolism, respectively.Therefore, both strains were not able to grow on M9 minimal medium supplemented with lactose or arabinose, respectively.Two pSwap plasmids (pSwap lacZ/pSwap araB) harbouring either the missing part of lacZ or araB (INS) flanked by corresponding homologous regions HA and HB as well as sgRNAs (T1), specific for the deletion region, were then assembled and transformed into the lacZ and araB knock-out strains.After induction of the CRISPR/Cas9 and λ RED systems, green and non-green colonies were spotted on M9 plates supplemented with either lactose or arabinose.Clones, which scarlessly integrated the missing coding sequence into their genome, were again able to grow on the corresponding saccharide.Clones were verified exemplarily by PCR and Sanger sequencing.For lacZ and araB reconstitution, green colonies attained an editing efficiency of up to 100% with low variation, while non-green colonies resulted in an editing efficiency of about 20% and 60% with a higher variation, respectively (see Fig. 4A).Additionally, the colony color distribution in different lacZ reconstitution experiments revealed a high variation of the green to non-green ratio (see Fig. 4B).In some experiments the number of green colonies dropped to a few counts on the whole plate whereas editing efficiency of green colony remained high.The frequency of green colonies depends on the timing of suppressor mutant emergence and subsequent outgrowth and is, therefore, random and not controllable.The earlier the mutation during growth, the higher the percentage of suppressors on the plate.Consequently, for some experiments, the total efficiency would drop to a low percentage, due to accumulation of suppressor mutants.However, within the pool of green colonies, the editing efficiency is significantly higher, very stable between replicates and not correlated to the frequency of green colonies.Hence, the multi-color system of SWAPnDROP increases and stabilizes the expected editing efficiency of CRISPR/Cas9 gene editing.Analogous to the in-vivo pSwap cleavage of the green-color selection for the Indel and Swap recombination, a red-color selection was implemented for the Drop recombination.The pDrop plasmid harbours a partly duplicated, non-functional mScarlet gene, which is cleaved by CRISPR/Cas9 during the Drop.The cleavage is mediated by the sgRNA TM (target mScarlet) located on the pSwap plasmid (see Fig. 2F).Homologous recombination of the overlapping sequences results in a functional mScarlet gene and therefore in the formation of red colonies (see Fig. 2F and Fig. S1C,D).For a detailed plasmid map of pDrop see Figure 3.

Fast Iterative Genome Editing Using Alternating Plasmids
The CRISPR SWAPnDROP system comes with two complementary versions of the pSwap plasmid: pSwap Cm R and Gm R .The first harbours a constitutively expressed I-SceI, a I-CreI recognition site and a Cm R cassette.The second harbours a constitutively expressed I-CreI, a I-SceI recognition site and a Gm R cassette (see Fig. S2).During recombination events in 7/26 the editing process, the meganuclease genes are removed from the pSwap plasmid by CRISPR/Cas9 excision together with the vioC gene (see Fig. 2D).The resulting green edited clones carry a pSwap' plasmid only harbouring the recognition site of the other meganuclease (see Fig. 2D).Transformation of a fresh pSwap plasmid cures the clone from the old pSwap by expression of the meganuclease located on the fresh pSwap and allows for another round of editing (see Fig. S2).Functionality was tested in E. coli MG1655 wild type for 12 consecutive genomic inserts at 10 distinct loci without problems in plasmid curing or evident functional loss of the editing system.During the functionality test, different recombination sites (FRT/loxP) as well as random DNA and origins of replication (F-plasmid oriS/native E. coli oriC) were inserted and replaced throughout the genome of E. coli MG1655 (see Fig. S5).The use of alternating pSwap plasmids facilitates one round of editing every 3 days.This is particularly useful in case of the absence of a suitable PAM site.A new site can be introduced by a first edit and the desired edit can be done efficiently in a consecutive edit.Moreover, the approach not only facilitates and speeds up consecutive rounds of edits, but also makes CRISPR SWAPnDROP compatible with automated editing approaches.

Multiplex Genome Editing
To assess the efficiency of CRISPR SWAPnDROP multiplex genome editing, the two E. coli genes, lacZ and araB, were reconstituted simultaneously.In a first step, an E. coli strain was generated by two consecutive gene knock-outs of lacZ and araB via CRISPR SWAPnDROP.Consequently, the strain was unable to grow on M9 plates supplemented with lactose or arabinose.In a second step, a pSwap for parallel repair was assembled.The pSwap plasmid contained the two repair templates (HAlacZ-INSlacZ-HBlacZ-TS-HAaraB-INSaraB-HBaraB) separated by an additional Cas9 excision site (TS) as well as the sgRNAs (T1, T2) targeting the non-functional lacZ and araB deletion sites (see Fig. S3).The repair templates were cloned via Golden Gate cloning in pHAIB using 6 PCR fragments (HAlacZ, INSlacZ, HBlacZ-TS, HAaraB, INSaraB, HBaraB).The additional Cas9 target site (TS) separates the templates for each locus of separate recombination via λ RED.After induction of CRISPR/Cas9 and λ RED, green colonies were transferred to M9 plates containing either lactose or arabinose.Consequently, only colonies with successfully reconstituted lacZ and araB genes were able to grow on both plates.The editing efficiency of the dual-repair approach was 98%, which means almost all clones restored their ability to grow on both saccharides.Even though, CRISPR SWAPnDROP is currently not able to generate more than two simultaneous edits due to its limitation of two sgRNAs, we wanted to assess the multiplex editing potential of larger numbers of parallel edits for future extensions of the system by sgRNA arrays.Therefore, two additional genes, xylA and dapA, were disrupted in the lacZ and araB double mutant.XylA encodes for a xylose isomerase and is essential for the xylose catabolism, while dapA encodes for a dihydrodipicolinate synthase, which is essential for cell wall synthesis.Disrupting those genes leads to a strain, which is DAP auxotroph and not able to use xylose as a carbon source.The resulting strain contained the four non-functional genes lacZ, araB, xylA and dapA.The lack of two sgRNAs was compensated by introducing the same Cas9 target sites for later repair in xylA and dapA knockouts as used for the lacZ and araB knockouts respectively.This allowed us to simultaneously modify four different loci, while only using two sgRNAs (T1, T2).In analogy to the double repair, the pSwap plasmid used for the reconstitution contained the four repair templates, each separated by an additional Cas9 target site (TS).Green colonies were tested on their ability to grow solely on lactose, arabinose and xylose each supplemented with DAP as well as on glucose without DAP.On average, 83% of the tested clones were able to grow on all saccharides as well as without DAP (see Fig. 4C).This shows the high editing efficiency of CRISPR SWAPnDROP even for up to four simultaneous edits and supports future extensions of the system by sgRNA arrays 18 .

Transfer of Large Chromosomal Regions
CRISPR/Cas9-mediated excision of double-stranded DNA and λ RED-mediated recombination generally permit the handling and alteration of large DNA fragments in vivo 16 .CRISPR SWAPnDROP extends the handling of large DNA fragments to a systematic excise and insert.Coupled with the modular assembled pSwap and pDrop plasmids, CRISPR SWAPnDROP provides a tool to rearrange the chromosome and transfer large chromosomal DNA between bacterial strains.As a proof of concept, we transferred and integrated a 151kbp chromosomal fragment from one E. coli strain into another.For the sequence to be transferred we chose a chromosomal region (del4), known to cause little impact on strain fitness upon deletion 34 (see Fig. S4).Using CRISPR SWAPnDROP, we first generated a E. coli knockout strain of the del4 region as acceptor for the region.For that, E. coli MG1655 was first transformed with cr3EC.A pSwap plasmid was assembled harbouring homologies HA and HB flanking the del4 region as well as a short random insert sequence, which eventually replaced the deleted region.Not containing the del4 region avoids possible toxic effects and homology issues due to an additional copy of the del4 region within the E. coli chromosome during and after transfer and facilitates transfer validation.After deletion, the E. coli ∆del4 was transformed with a pDrop plasmid (pDrop del4) harbouring the sgRNA for selection upon reintegration.A pSwap (pSwap del4) was then assembled and transformed into the E. coli RP4 conjugation strain MFDpir to load the del4 region onto the plasmid.The del4 region of E. coli MFDpir is about 151kb in size and a bit smaller than the deleted E. coli MG1655 del4 region due to a ˜16kb deletion within the region.The loaded pSwap' plasmid was conjugated to E. coli ∆del4 and the 151kbp region was integrated into the ∆del4 chromosomal region (E.coli∆del4::MFDdel4).Colony pcr revealed an integration efficiency of up to 60% and on 8/26 average around 40% (45 clones were tested).Then, pcr and sanger sequencing was used for verification of each strain.As seen in Fig. 5, we were able to amplify DNA from the del4 region in E. coli MG1655 as well as in E. coli∆del4::MFDdel4, while in E. coli ∆del4 those bands did not appear on the electrophoresis gel.The 3kb PCR band spanning the del4 region appeared only in E. coli ∆del4, as for the strains still harbouring the del4 region the PCR product would have been above 167kbp.To distinguish between E. coli MG1655 and E. coli∆del4::MFDdel4, homologies Hα and Hβ were chosen to delete small regions flanking the del4 region after integration.DNA bands of lower size from PCRs spanning this small deleted region indicate the integration of the MFDdel4 region into the ∆del4 mutant chromosome (see Fig. 5A).To rule out that specific bands originate from the pSwap' plasmid, the strain was tested phenotypically and via PCR for the loss of the plasmid.The strain was unable to grow on the corresponding antibiotic and no plasmid specific amplicons were detected.E. coli∆del4::MFDdel4 is a chimera of the MG1655 and the E. coli MFDpir at the del4 locus.To verify the chimeric nature of the strain, we performed next-generation sequencing (NGS) data of the E. coli∆del4::MFDdel4 (see Fig. 5B).NGS reads of E. coli∆del4::MFDdel4 and E. coli MG1655 were mapped against the E. coli MG1655 reference genome.While for E. coli MG1655, the del4 region is 167kbp in size, about 16kbp are missing in the del4 region of E. coli MFDpir.Consistent with a successful edit, reads of the 16kbp deletion as well as the deleted del4 flanking regions were missing, while reads of the complete MFDdel4 region were present.

Interspecies Gene Transfer and Genome Editing in Vibrio natriegens
The fast-growing gammaproteobacterium V. natriegens has the potential to rise as an important organism in biotechnology and molecular biology.In recent years, this organism was made genetically accessible, but still lacks a working CRISPR/Cas9-based gene editing system 35,36 .Using MoCloFlex, we adapted CRISPR SWAPnDROP to V. natriegens.In contrast to the pSwap plasmid, the cr3Ec plasmid was not compatible with V. natriegens.Hence, we generated a new cr3 version called cr3Vn.To test the system, a pSwap plasmid (pSwap recJ) was assembled containing the selective sgRNA (recJ) and homology arms HA and HB of ∼1.2kb and ∼1.4kb in length, which flank the exonuclease gene recJ.Inactivation of recJ was shown to enhance natural transformation in other Vibrio species 37 .Therefore, the recJ locus was chosen with regard to possible future cloning applications.V. natriegens was transformed with both cr3Vn and pSwap recJ and purple colonies were used for induction of the Cas9 and λ RED genes.Subsequent colony PCR and sanger sequencing revealed recJ knock-out clones in 23% of the tested clones (3 out of 13).After showing editing activity of CRISPR SWAPnDROP in V. natriegens, we systematically determined the editing efficiency and optmized the induction protocol (see Material and Methods).In analogy to the quantification of the editing efficiency in E. coli, we intended to use a lacZ knockout repair approach.For that, we needed a knockout lacZ variant of a functional lac operon.As V. natriegens has no functional lac operon, a native lacZ knockout was no option 38 .Consequently, we planed to transfer the E. coli ∆lacZ variant used for E. coli to V. natriegens.First, we transferred the native E. coli lac operon to V. natriegens to test its functionality.For the transfer, we assembled a pSwap plasmid with homology arms (H1α, H2β ) and sgRNAs (T1, T2) flanking the E. coli lac operon (pSwap lacVn).The lac operon was loaded onto the pSwap' and transferred to V. natriegens via conjugation.Without additional adaptations to the lac operon, V. natriegens was able to process X-gal to form blue colonies upon induction with IPTG.In the next step, we transferred the lac operon of the E. coli ∆lacZ knockout strain to V. natriegens harbouring cr3Vn and pDropVn recJ and dropped it into the recJ chromosomal locus.For the selective sgRNA of pDropVn recJ, the spacer sequence of the initial recJ knockout test was used.For the transfer, we reused pSwap lacVn initially designed to allow for a drop into the recJ of V. natriegens.Integration was verified by PCR and Sanger sequencing of the transitional region of the insert fragment and the flanking chromosomal region.pSwap plasmid elimination was verified phenotypically by the inability to grow on corresponding antibiotics.With the resulting V. natriegens lac Ec ∆lacZ strain, repair was performed using the lacZ repair pSwap (pSwap lacZ) already used in E. coli genome editing efficiency determination.Green and white colonies were examined for their ability to form blue color upon growth with IPTG and X-gal, which implies a successful editing event.The editing efficiency of the lacZ repair in V. natriegens was up to 92% and on average around 65% (see Fig. 8).Interestingly, no non-green colonies appeared blue upon growth with IPTG and X-gal indicating an enhanced editing efficiency of green colonies also in V. natriegens.Additionally, we tested alpha-complementation in V. natriegens.In analogy to the previous lac operon transfer, we transferred the lac operon of DH5α harbouring only the lacZ ω fragment, capable of alpha-complementation.After transfer and integration into the recJ locus by CRISPR SWAPnDROP, the strain was transformed with PICH41308 19 , constitutively expressing the lacZ α fragment.Blue colonies on plates containing IPTG and X-gal indicated a successful α-complementation (see Fig. 7A).Finally, we tested the transfer between species and subsequent integration of a large DNA fragment.For this purpose, we transferred the 151kb E. coli MFDdel4 region to V. natriegens and integrated it into the recJ chromosomal locus.Analogous to the swap and drop of this region between two E. coli strains, we first assembled the same pSwap as for the E. coli swap, but exchanged the Hα and Hβ regions (pSwap del4Vn).Afterwards, the MFDdel4 region was loaded onto the pSwap plasmid in E. coli and subsequently conjugated to the V. natriegens strain harbouring cr3Vn and pDropVn recJ.After dropping the 151kb region into the recJ locus of V. natriegens, colony PCR revealed an efficiency of 90%.Plasmid specific PCR and the inability to grow on corresponding antibiotics indicated the elimination of the pSwap' plasmid.As seen in Fig. 6A, pcr from V. natriegens recJ::MFDdel4 showed DNA bands of the del4 region 9/26 (see Fig. 6A "1-6") as well as of the transition between the del4 and the adjacent chromosomal regions (see Fig. 6A "8-9").Additionally, amplification of the recJ locus was no longer possible (see Fig. 6A"7").In order to verify the integrity of the region, we performed next-generation sequencing (NGS).NGS reads of V. natriegensrecj::MFDdel4 and V. natriegens WT (ATCC 14048) were mapped against the V. natriegens recj::MFDdel4 reference genome.Data analysis revealed that indeed reads for the complete 151kbp MFDdel4 region were present in the recJ locus of V. natriegens recj::MFDdel4, while they were absent in V. natriegens WT (see Fig. 6B).

The Escherichia coli Lac Operon Enables Lactose Utilization and Strongly Improves Galactose Utilization in Vibrio natriegens
The V. natriegens lac Ec strain created during editing efficiency determination was able to process X-gal.Therefore, we further analysed the lactose metabolism.Growth experiments indicated that V. natriegens lac Ec was able to grow on M9 minimal medium with lactose as its sole carbon source (see Fig. 7B,C), even with a slightly higher growth rate (µ= 0.67 h −1 ) compared to growth on glucose(µ= 0.50 h −1 ).A similar phenomenon was observed for the disaccharide sucrose compared to glucose 38 .Growth rates on rich medium and glucose were slightly reduced compared to wild type.This was also observed in pure ∆recJ strains of the initial knockout experiments.Utilization of the second downstream product of lactose degradation, galactose, was strongly improved (µ= 0.59 h −1 ) compared to wild type (µ= 0.08 h −1 ).In order to exclude secondary mutations in V. natriegens lac Ec as a source of these changes, we removed the lac operon using CRISPR SWAPnDROP.The resulting ∆lac Ec strain showed the loss of lactose metabolization and the strongly reduced wild type level of galactose utilization 38 .It retained the slightly reduced growth rates on glucose and rich medium of a recJ mutant.Hence, the improved galactose utilization is directly connected to the presence of the E. coli lac operon.The mechanism of action is yet unknown and should be investigated in future studies.

Discussion
With CRISPR SWAPnDROP we present a versatile scarless and marker-free genome editing system independent of size and with high editing efficiencies.With its multi-color co-selection system we introduce a novel approach to co-selection not relying on chromosomal insertions or other persistent scars and at the same time increasing editing efficiencies and monitoring the assembly process of the modular CRISPR SWAPnDROP system.With its high editing efficiency of above 90% for E. coli, CRISPR SWAPnDROP matches the current CRISPR/Cas9-based editing systems 14,15,39 and integrates desirable features of several systems with high flexibility (see table 6).CRISPR SWAPnDROP is also capable of multiplexed genome editing.We could show, that even with four parallel edits, the editing efficiency remains well above 80%.This suggests the integration of sgRNAs arrays that have shown to perform up to 30 parallel edits 17,18 in future extensions.Its modular design renders integration of these arrays possible without alteration of essential concepts e.g. by replacing the T1 and T2 modular plasmids by a sgRNA array plasmid.Another aspect of the modular design is the application of libraries for chromosomal integration.In the insert plasmid INS, sequence libraries can be cloned resulting in a broad variety of inserts for a specific location.Furthermore, CRISPR SWAPnDROP is compatible with current cloning systems such as MoClo, MoCloFlex and the Marburg Collection 19,20,22 .This grants access to already established large DNA libraries and facilitates integration of CRISPR SWAPnDROP to present library systems.DNA libraries can be applied in protein modification or pathway optimization and can be directly tested at native chromosomal loci without intermediate plasmid constructs.The modular assembly of the key plasmids cr3 and pSwap simplifies adaptation to other species and facilitates updates with new Cas or recombination systems 40,41 .Using MoCloFlex 20 and Marburg collection 22 systems, only a few modular plasmids carrying resistance cassettes and induction systems needed to be modified to adapt CRISPR SWAPnDROPs' cr3 for V. natriegens.Furthermore, cr3 can potentially be modified to support genome editing in other species with a different recombination system and Cas9 activity.It has been shown, that Cas9 is active in all kingdoms of life including eukaryotes, fungi, plants, bacteria and archea.Therefore, we can assume Cas9 activity in most species.Recombination systems are more species specific.Therefore, for each species, a functional recombination system has to be present.Such systems can usually be found in species-specific phages or in the organism itself.Such recombination systems were also found in all kingdoms of life [42][43][44] and are widely used for genome editing.Furthermore, present recombination systems can be applied in other species by additional expression of missing or incompatible components like single-stranded DNA-binding proteins (SSB) 45 .Therefore, suitable recombination systems should be available for most species.The modular design of CRISPR SWAPnDROP is based on the type IIS restriction enzymes BbsI and BsaI.Therefore, homology arms and inserts should be free of these sites to be applied in the system.The recognition sites of the applied restriction enzymes are 6 bases long and therefore occur on average every 4096 bp.For optimal recombination efficiency, homology arms should be about 500 bp in E. coli 27 and about 1000 bp for V. natriegens 28 .However, it is possible to reduce the length to 50 bp for E. coli and 200 bp for V. natriegens.Therefore, the frequency of a restriction site in a homologous arm is statistically about 1/10 for V. natriegens and 1/20 for E. coli.This, however, may cause a reduction in editing efficiency.Alternatively, restriction sites can be preserved, especially within inserts, by adding a final ligation step to the Golden Gate reaction protocol to religate the preserved sites (see Material and Methods).It is apparent, that the additional restriction sites reduce assembly efficiency.For cloning of homologies and insert parts, usually one to three fragments are assembled.Within this range, efficiency of Golden Gate cloning is extremely high, especially with ccdB/lacZα counter-selection.Therefore, a reduced efficiency is no practical burden.For the seven parts of the pSwap assembly, the violacein color system provides efficient selection for proper clones.Cloning efficiency can be further improved by transformation of a cloning strain instead of the strain of interest.A general problem of Cas9-based counter-selection is the dependence on PAM sites (NGG) at the locus of interest.Although PAM sites occur on average every 16 bp, in some cases the PAM site might not be situated at the perfect location for a scarless edit.In this case, an additional edit needs to be performed to introduce a PAM site at the desired location.With this new PAM site, the actual scarless edit is possible in a second step.With its ability for stable iterative rounds of genome editing, CRISPR SWAPnDROP facilitates such consecutive edits.In this study, we have shown the stability of the system in 12 consecutive edits.Therefore, the system can be potentially applied in automated cloning approaches 33 .The application of the meganucleases I-SceI and I-CreI to cure plasmids from the previous iteration, restricts this method to organisms without naturally occuring sites for these meganucleases.However, the length of 18bp and 22bp for I-SceI and I-CreI practically almost excludes occurances of such sites.The sites occur on average every 68 Gb for I-SceI and every 17 Tb for I-CreI.To illustrate this: Even in the 1000 fold larger human genome no such site is present.However, for the rare case of an occurance in the genome, the meganucleases and its site on the H2 plasmids can be modified to avoid undesired restriction 46,47 .The multi-color selection system introduced in this study improves editing efficiency by selecting out the major part of suppressor mutants that are in previous concepts part of the clones screened for successful edits.Its mechanism provides quality control for the recombination and restriction systems involved in the edit.In general it relies on parallel plasmid-born edit with a clear phenotype.This concept is realised by Cas9 mediated vioC removal and subsequent recombination to reconstitute the pSwap plasmid.Only if Cas9 restriction and recombination are functional green colonies appear on the plate.Therefore, green colonies have a higher chance to yield successful edits and results between replicates are more stable.Like any other selector, the multi-color system cannot filter all suppressor mutants (e.g.mutations on the target site or sgRNA mutations), therefore editing efficiency is below 100% and residual variance between replicates is detected.In comparison to E. coli, V. natriegens editing efficiency is slightly lower.The decrease in editing efficiency may lie in the efficiency of λ RED.The recombination system is evolutionary optimized for E. coli and apart from the induction system, we made no attempts to further optimize codon usage or RBS strength for λ RED in V. natriegens.An optimized expression of λ RED may improve efficiency even more.Still, screening of two colonies yields a positive clone on average.Therefore CRISPR SWAPnDROP is highly effective in V. natriegens.CRISPR SWAPnDROP is capable of transferring large chromosomal regions between strains and species.We tested the system in Hfr+ strains.By adding an RP4 plasmid derivative or by triparental mating, the approach can in principle be extended to any strain of interest.RP4 is a broad-host-range conjugation system, functional in gram-negative bacteria 48 and various gram-positive bacteria 49 .Large scale DNA transfer of CRISPR SWAPnDROP can be applied in synthetic chromosome construction for biotechnology and basic research 29,30,50 .The technical and human effort for the in-vitro assembly of synthetic chromosomes strongly increases with its size 19 .In addition, with size, the chance for type IIS restriction sites within the DNA of interest rises.In the 6kb lac operon, already 4 natural BbsI and 3 Esp3I sites usually used in modular cloning systems are present.In the 151kb region 37 BbsI, 8 BsaI and 37 Esp3I are present.Hence, at a certain size of the construct, either site removal 31 or homology based methods are required.Similar to modular cloning, assembly approaches based on yeast recombination suffer from laborious and time consuming transfer back and forth between yeast and the organism of interest via in-vitro methods 51 .With an iterative approach using modular assembly at the small scale to design customized parts and CRISPR SWAPnDROP for the assembly of the smaller parts to large chromosomes technical size limitations can be overcome and costs could be reduced drastically.With the transfer of 151kb of DNA at an efficiency above 40%, we have shown that CRISPR SWAPnDROP is not limited in size within the limits of species-specific chromosome plasticity.Additionally, the presented transfer of the E. coli lac operon into V. natriegens is an example for a successful gain of function genome edit.Hence, it is possible to introduce desired properties from different organisms or a DNA library to construct tailor made organisms.Next-Generation Sequencing of E. coli mutant after 12 consecutive edits.Shown is the next-generation sequencing (NGS) coverage of a E. coli mutant strain after 12 consecutive edits (light grey) compared to the wild-type E. coli MG1655 (dark grey).For the mutant strain, several recombination sites (FRT/loxP) as well as random DNA and origins of replication were inserted into the chromosome using iterative CRISPR SWAPnDROP genome editing.Mutant and wild-type E. coli NGS reads were aligned against the mutant reference genome and the sectors of each edited site (A-J) as well as the complete genome coverage (circle) are shown.Reads at all insertion locations (dashed lines) are present for the mutant strain, while no reads are present for the wild-type strain (red rectangle).(A) and (E) show the coverage of a chromosomal location after 2 different edits at the same position.For (A), the native origin of replication oriC of E. coli was first replaced with the F-plasmid derived oriS origin of replication.oriS was then replaced again with oriC while introducing scar sites flanking the oriC.Scar sites were introduced due to using HA, INS and HB plasmids instead of HAIB plasmid.The absence of those flanking scars in the wild-type are highlighted with red rectangles in (A).For (E), oriC was first inserted into the insPQ locus and then replaced with a random sequence.The absence of the random sequence in the wild-type strain is highlighted with a red square.

Figure 3 .Figure 4 .
Figure 3. Detailed map of the plasmids cr3Ec, pSwap and pDrop.The outer layer contains relevant features of each plasmid.The white inner layer (if applicable) represents the modular parts prior to assembly using MoCloFlex (MCF) or the CRISPR SWAPnDROP assembly system.The black inner layer (if applicable) shows the location of variable elements (orange) of the plasmids specific for each edit.Cas9 and meganuclease target sites are indicated by scissors.The sgRNA loci and the respective target sites share the same color.

Figure 5 .
Figure5.Integration of a 151kbp chromosomal region via CRISPR SWAPnDROP.Shown are agarose gels of PCRs from the transfer and integration of a 151kbp chromosomal region in E. coli.PCRs were done for E. coli MG1655, the acceptor (E.coli∆del4) and the edited strain (E.coli∆del4::MFDdel4).PCRs "1-6" result in the amplification of fragments of the del4 region evenly distributed over the 167kbp(E.coli MG1655) and 151kbp (E. coli MFDpir), respectively.PCR "7" result in the amplification of a 3000bp fragment, if the del4 region is missing.PCR "8" and "9" amplify the transition of the del4 region to the adjacent chromosomal regions.Upon integration of the 151kb region into the E. coli∆del4 chromosome, small regions flanking the 151kbp region were deleted resulting in DNA bands of lower size compared to the WT chromosome.This is additionally highlighted with the colors green and red.Dashed line indicates the band size of a 2000bp DNA fragment for a better comparison.(B) Shown is the next-generation sequencing (NGS) coverage (reads/base) of E. coli∆del4::MFDdel4 (light grey) and E. coli MG1655 (dark grey) mapped against the E. coli MG1655 reference genome.The middle plot shows the coverage of the del4 region.For E. coli∆del4::MFDdel4, reads for the 16kbp deletion, only present in the MFDdel4 region, are missing (red rectangle).The upper and lower plots showing the detailed coverage of the del4 flanking regions.Upon integration of the MFDdel4 region, small flanking regions were deleted, shown as missing reads between the dashed lines.

Table 1 .
Primer overhangs and antibiotics used for pSwap parts construction Lower-case and red letters indicate the BsaI restriction site and the required overhangs, respectively.Underlined letters represent the Cas9 cut site (TD) and have to be added to the primer if homology α and β are used.

Table 2 .
Plasmids and corresponding antibiotic resistance for E. coli

Table 3 .
Plasmids and corresponding antibiotic resistance for V. natriegens