# CRISPR SWAPnDROP – A multifunctional system for genome editing and large-
scale interspecies gene transfer

Marc Teufel  Philipps Universität Marburg, Synthetic Microbiology Center
Marburg (SYNMIKRO), Marburg, 35043, Germany  Carlo A. Klein  Philipps
Universität Marburg, Synthetic Microbiology Center Marburg (SYNMIKRO),
Marburg, 35043, Germany  Maurice Mager  Philipps Universität Marburg,
Synthetic Microbiology Center Marburg (SYNMIKRO), Marburg, 35043, Germany
Patrick Sobetzko  Philipps Universität Marburg, Synthetic Microbiology Center
Marburg (SYNMIKRO), Marburg, 35043, Germany<EMAIL_ADDRESS>marburg.de

###### Abstract

The need for diverse chromosomal modifications in biotechnology, synthetic
biology and basic research requires the development of new technologies. With
CRISPR SWAPnDROP, we extend 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 and the currently fastest
growing and biotechnologically relevant organism Vibrio natriegens. We
demonstrate the excision, transfer and integration of 151kb chromosomal DNA
between E. coli strains and from E. coli to V. natriegens without size-
limiting intermediate DNA extraction. With the transfer of the E. coli MG1655
wild type lac operon, we establish a functional lactose and galactose
degradation pathway in V. natriegens to extend its biotechnological spectrum.
We also transfer the E. coli DH5${\alpha}$ lac operon and make V. natriegens
capable of ${\alpha}$-complementation - a step towards an ultra-fast cloning
strain. Furthermore, CRISPR SWAPnDROP is designed to be the swiss army knife
of genome engineering. Its spectrum of application comprises scarless, marker-
free, iterative and parallel insertions and deletions, genome rearrangements,
as well as gene transfer between strains and across species. The modular
character facilitates DNA library applications and the recycling of
standardized parts. Its novel multi-color scarless co-selection system
significantly improves editing efficiency to 92% for single edits and 83% for
quadruple edits 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 $\lambda$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 specificity is mediated
by a guide RNA (gRNA) complimentary to the target sequence. Possible target
sites are only limited by the protospacer adjacent motive (PAM) sequence
(5’-NGG-3’), which is required to be located upstream of the target sequence.
Due to its simplicity, Cas9 counter-selection is widely used. Plasmid systems
harbouring an inducible Cas9 and a recombination system were designed to
select for gene deletions, point mutations and short insertions[14, 15]. These
systems depend on the transformation of synthetic oligonucleotides or linear
double-stranded template DNA for homologous recombination and are therefore
not suitable for large-size insertion. To overcome size limitations, REXER
provides the template DNA on an episomal replicon [16]. Inserts of up to 100kb
are excised in vivo by CRISPR/Cas9 to facilitate $\lambda$RED recombination.
Selection occurs via positive and negative selection markers, which leads to
scars at the integration site and requires a previous integration of the the
first selection-marker set. For modularization and increased flexibility of
CRISPR/Cas9 based methods modular cloning techniques like Golden Gate Cloning
have been employed for sgRNA arrays [17, 18]. Furthermore, joining of insert
and homology arms as well as the preparation of the locus specific protospacer
require an additional cloning step favouring the concept of modular cloning.
Independent of the CRISPR/Cas9 methods, general approaches for modular cloning
were developed to assemble larger constructs from small standardized parts
e.g. MoClo, MoCloFlex, Golden Mutagensis and Marburg Collection[19, 20, 21,
22]. However, modular cloning for CRISPR/Cas9 editing has not yet been
systematically implemented. The flexibility of modular systems comes at a
price. The sequence integrity of the parts from verified plasmid-born DNA
libraries is highly stable and needs no additional sequencing after assembly.
However, the absence of individual parts by incorrect ligation order poses a
permanent thread to the functional integrity of the final construct. Here we
present CRISPR SWAPnDROP, a versatile genome editing system for bacteria.
Following a modular concept, CRISPR SWAPnDROP includes a scarless and marker
free system for large-scale insertions, deletions and in-vivo chromosomal DNA
transfer between strains and even species. For compatibility with automated
genome editing, it is capable of multiplexing and iterative genome
modifications. Its multi-color selection system efficiently avoids errors in
system assembly and provides a scarless co-selection system for increased
editing efficiency.

## Material and Methods

### Strains, Media and Reagents

Escherichia coli MG1655 was used for all genome editing experiments in E.
coli, while E. coli DH5${\alpha}$ was used for cloning purposes. For
conjugation experiments a precursor strain of MFDpir harbouring a stable RP4
conjugation machinery and additionally modified with dapA and endA knockouts
was utilized [23]. All E. coli strains were cultivated in LB (10g/L tryptone ,
5g/L yeast extract, 10g/L NaCl) at 37°C and 200rpm shaking. If needed, LB was
supplemented with anhydrotetracyclin3 (100 ng/ml), arabinose (1%) or 300µM
DAP. For experiments requiring minimal medium, M9 medium (33.7mM Na2HPO4; 22mM
KH2PO4; 8.6mM NaCl; 9.4mM NH4Cl; 1mM MgSO4; 0.3mM CaCl2) supplemented with
different carbon sources (lactose, arabinose, xylose, glucose: 0.5%) was used.
For cloning, Q5 HF Polymerase (NEB), BsaI-HFv2 (NEB) and BbsI (NEB) were used.
In this study, experiments with V. natriegens were carried out in V.
natriegens ATCC 14048 ${\Delta}$VNP12[24]. V. natriegens was cultivated in LB
supplemented with V2 salts (204mM NaCl, 4.2mM KCl, 20.14mM MgCl2) (LBv2) at
37°C and 200rpm shaking. For growth of V. natriegens in minimal medium, M9
medium supplemented with with different carbon sources (0.4%) and additional
2% NaCl was used. Preparation of chemically competent cells and transformation
was carried out according to Green and Rogers[25] and Stukenberg et al.[22]
for E. coli and V. natriegens, respectively.

### 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);
H1${\alpha}$, H2${\beta}$, 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
($\sim$25ng/1000bp) of PCR products were utilized for cloning. Annealed
oligonucleotides or PCR products were mixed with 40fmol of the corresponding
entry vectors, 1X T4-Ligase buffer (NEB), 1µl (20 units) BsaI-HFv2 (NEB), 1µl
(400 units) T4-Ligase (NEB) and MilliQ water to a final volume of 20µl. The
mix was incubated for 2-5 hours at 37°C for in vitro assembly. In case of
internal BsaI sites in the inserts, a final 30 minutes ligation step at 16°C
prior to heat inactivation is added for optimal Ligase activity and reduced
BsaI activity. The mix was transformed into chemically competent DH5${\alpha}$
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, H${\alpha}$\- and H${\beta}$
homologies for H1${\alpha}$ and H2${\beta}$ 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, H1${\alpha}$, H2${\beta}$, 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 (DH5${\alpha}$) 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 H2${\beta}$ (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 H1${\alpha}$ and H2${\beta}$ (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.

Table 1: Primer overhangs and antibiotics used for pSwap parts construction

Part | FW Primer overhang | REV Primer overhang | Antibiotic
---|---|---|---
| (5’-overhang) | (5’-overhang) | (µg/ml)
T1 | GAggtctcAGCAC | GAggtctcAAAAC | kanamycin (35)
T2 | GAggtctcAGCAC | GAggtctcAAAAC | kanamycin(35)
H1${\alpha}$ | GAggtctcAGGAG- | GAggtctcAACGG | kanamycin(35)
| GCTCGATCGCTAATCGTTATCGG |  |
H2${\beta}$ | GAggtctcAGGAG | GAggtctcAAGCG- | chloramph.(18)
|  | GCTCGATCGCTAATCGTTATCGG | gentamycin (10)
HA | GAggtctcACCGT | GAggtctcACTCC | kanamycin(35)
HB | GAggtctcACCGT | GAggtctcAAGCG | kanamycin(35)
INS | GAggtctcAGGAG | GAggtctcAAGCG | kanamycin(35)
HAIB | GAggtctcACCGT | GAggtctcAAGCG | kanamycin(35)

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 ${\alpha}$ and ${\beta}$ are
used.

### 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 Ptet repressor (TetR) needed to
be transformed first to establish Ptet 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 ${\Delta}$VNP12 was first transformed with
cr3Vn. Subsequently, this strain was transformed with pSwap CmR and incubated
on LBv2 Agar with corresponding antibiotics at 37°C over night (see Table3).
The plates were incubated at room temperature for several hours to develop its
purple color. Purple colonies were inoculated and grown until OD600 reached 4
or over night in LBv2 with appropriate antibiotics at 37°C and 200rpm shaking.
Induction of the $\lambda$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 ${\Delta}$dapA ${\Delta}$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 OD600 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 $\lambda$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
OD600 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
$\lambda$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.

Table 2: Plasmids and corresponding antibiotic resistance for E. coli Plasmid | Antibiotic resistance (µg/ml) | Additional information (Inducer)
---|---|---
cr3Ec | Kanamycin (35) | aTet/Arabinose (Cas9/$\lambda$RED)
pSwap CmR | Chloramphenicol (18) | aTet (sgRNA)
pSwap GmR | Gentamycin (10) | aTet (sgRNA)
pDrop | Ampicillin (100) | aTet (sgRNA)
Table 3: Plasmids and corresponding antibiotic resistance for V. natriegens Plasmid | Antibiotic resistance (µg/ml) | Additional information (Inducer)
---|---|---
cr3Vn | Kanamycin (200) | aTet/Arabinose (Cas9/$\lambda$RED)
pSwap CmR | Chloramphenicol (2) | aTet (sgRNA)
pDropVn | Gentamycin (50) | aTet (sgRNA)

## 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 (H$\alpha$, H$\beta$) 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, 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 H${\alpha}$ and H${\beta}$
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, H${\alpha}$, H${\beta}$),
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 lacZ${\alpha}$ 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 (CmR, GmR) 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, H1$\alpha$ 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 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 $\lambda$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 (Ptet), 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 $\lambda$RED system was used. Its transcription
unit was assembled using the promoter part harbouring the PBAD 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 [32, 28]. In this study, we modified
the E. coli $\lambda$RED operon by setting it under the control of the native
V. natriegens PBAD 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 ${\beta}$-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 $\lambda$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 CmR and GmR. The first harbours a constitutively expressed
I-SceI, a I-CreI recognition site and a CmR cassette. The second harbours a
constitutively expressed I-CreI, a I-SceI recognition site and a GmR cassette
(see Fig. S2). During recombination events in 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 $\lambda$RED. After induction of CRISPR/Cas9 and
$\lambda$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 $\lambda$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 ${\Delta}$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 ${\Delta}$del4 and the 151kbp region was integrated into the
${\Delta}$del4 chromosomal region (E. coli${\Delta}$del4::MFDdel4). Colony pcr
revealed an integration efficiency of up to 60% and on 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${\Delta}$del4::MFDdel4, while
in E. coli ${\Delta}$del4 those bands did not appear on the electrophoresis
gel. The 3kb PCR band spanning the del4 region appeared only in E. coli
${\Delta}$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${\Delta}$del4::MFDdel4, homologies H${\alpha}$ and H${\beta}$ 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 ${\Delta}$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${\Delta}$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${\Delta}$del4::MFDdel4 (see Fig. 5B). NGS reads of E.
coli${\Delta}$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 $\sim$1.2kb and $\sim$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 $\lambda$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 $\Delta$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 (H1$\alpha$, H2$\beta$) 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 $\Delta$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 lacEc
$\Delta$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 DH5$\alpha$ harbouring only the
lacZ $\omega$ 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 $\alpha$
fragment. Blue colonies on plates containing IPTG and X-gal indicated a
successful $\alpha$-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 H${\alpha}$ and
H${\beta}$ 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
(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 lacEc 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 lacEc 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 $\Delta$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 lacEc as a source of these
changes, we removed the lac operon using CRISPR SWAPnDROP. The resulting
$\Delta$lacEc 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, 39, 15] 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 [18, 17] 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/lacZ$\alpha$ 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 $\lambda$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 $\lambda$RED in V.
natriegens. An optimized expression of $\lambda$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 [50,
29, 30]. 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.

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## Acknowledgements

The authors thank Kristala L. Jones Prather for providing Cas9 plasmids, Eric
Ellenberger and Werner Sobetzko for his help with photographs, Torsten
Waldminghaus for providing strains, Daniel Stukenberg for his help with the
Marburg Collection library and Mona Bastian, Sabrina Steidl and Antje Schäfer
for material and mental support. The project was funded by Deutsche
Forschungsgemeinschaft (DFG) [SO 1447/1-1, SO 1447/3-1 and SO 1447/5-1].

## Author Contributions Statement

M.T., C.K. and P.S. conceived the experiments. M.T., C.K., M.M. and P.S.
conducted the experiments. M.T., C.K., M.M. and P.S. analysed the results.
M.T. and P.S. wrote the manuscript. All authors reviewed the manuscript.

Day | Expenditure of time | Task
---|---|---
1 | 2 h | PCR (homologies, insert and targets), cloning, transformation in cloning strain
2 | 1 h | Colony screening and over-night culture
3 | 1 h | Plasmid preparation and sequencing
4(1) | 1 h | Assembly of pSwap and transformation in destination strain
5(2) | 5 min | Purple colony induction in liquid medium
6(3) | 5 min | Plating of over-night culture
7(4) | 1 h | PCR screening of green colonies and sequencing
8(5) | 1 h | Conjugation into the acceptor strain and plating
9(6) | 5 min | Green colony induction in liquid medium
10(7) | 5 min | Plating of over-night culture
11(8) | 1 h | PCR screening of red colonies and sequencing
Table 4: Workflow of CRISPR SWAPnDROP. The numbers in brackets are the days using a present DNA library. Gene editing and gene transfer share the same protocol until day 7(4) (Black line). The workflow for the gene transfer continues at day 8(5). System state | Cas9 RED | CRISPR SWAPnDROP
---|---|---
fully functional | white colonies | green colonies
Cas9 defect | white colonies | violet colonies
RED defect | no colonies | no colonies
deletion in plasmid | white colonies | white colonies
Table 5: Comparison of Cas9 RED based systems and CRISPR SWAPnDROP regarding suppressor detection. For the ’system state’ column a green or red color indicates the presence or absence of successful editing events, respectively. For the other columns, green or red color indicates the positive or negative expectation for observed colonies Feature | SWAPnDROP | REXER | RED | CRMAGE | No-Scar
---|---|---|---|---|---
scarless | + | + | - | + | +
marker-free | + | - | - | + | +
indels | + | + | + | + | +
large fragments | + | + | - | - | -
rearrangements | + | - | - | - | -
DNA transfer | + | - | - | - | -
modular | + | - | - | - | -
iterative | + | + | (+) | + | +
multiplex | + | - | - | + | -
Table 6: Feature comparison of current systems. A plus symbol indicates the
capability of the system. A plus in brackets indicates a conditional
capability e.g. in combination with other subsequent methods. A minus sign
indicates the lack of the feature. Figure 1: Overview and mechanism of CRISPR
SWAPnDROP. CRISPR SWAPnDROP is a genome editing system based on CRISPR/Cas9
counter-selection, homologous recombination and a multi-color scarless co-
selection. It facilitates scarless insertions and deletions (A) and is capable
of transferring chromosomal DNA between organisms (B + C). The pSwap plasmid
harbours the template for homologous recombination including the homologous
regions (HA, HB) and the insert DNA (INS), the sgRNA (TS) expression cassettes
for the excision of the double-stranded donor template and for the chromosomal
counter-selection (T1, T2). The excision is catalysed by CRISPR/Cas9
(Scissors), expressed from the helper plasmid cr3 together with the genes for
efficient homologous recombination. For the transfer of chromosomal regions,
chromosomal donor DNA (yellow) is additionally excised (T1, T2). The two
regions are swapped resulting in a pSwap plasmid loaded with a chromosomal
region of interest (pSwap’). The loaded pSwap’ plasmid can then be conjugated
to a cell harbouring cr3 and the second helper plasmid pDrop, which catalyses
the excision of the donor DNA on pSwap’ (TD) and the subsequent integration
into the acceptor chromosome via counter-selection (T3). Cell border color
represents the multi-color co-selection system. Cells change their color from
purple to green and from green to red after the Indel/Swap and Drop events,
respectively. Figure 2: pSwap assembly and recombination overview. CRISPR
SwapnDrop uses a modular assembly system for the pSwap construction. It
consists of seven modules (five for scarless integration) harbouring sgRNA
expression cassettes for target sites (T1,T2), homologous regions (H$\alpha$,
H$\beta$, H1, H2, HA, HB) and insert DNA (INS). (A) Each of those parts can be
cloned into a different entry vector using the type-IIS restriction enzyme
BsaI. (B) Each of those seven (five for scarless integration) modules are used
to assemble the pSwap plasmid. The assembly takes place in a one-pot, one-step
reaction using the type-IIS restriction enzyme BbsI. Therefore, it is possible
to combine and recycle different modules for the desired approach. (C) The
correct assembly of the pSwap plasmid is ensured by the expression of the
biosynthetic pathway of deoxyviolacein. Each of the fragments (T1, T2,
H1$\alpha$, HA) harbour parts of the deoxyviolacein expression cassettes. Only
if assembled correctly, production of deoxyviolacein is possible. Colonies
appear purple after transformation. (D) During linearization of the pSwap
plasmid by sgRNA TS and Cas9, the vioC gene is removed resulting in the
formation of prodeoxyviolacein after successful recombination. Colonies then
appear green. Additionally, the tool box comprises two pSwap plasmids
(CmR/GmR) harbouring either the I-SceI or I-CreI meganuclease genes and the
opposite recognition site. The removal of the meganuclease genes during
recombination allows iterative use of the pSwap plasmids and therefore
consecutive genomic edits. (E) Shown is the biosynthetic pathway of
L-tryptophan to deoxyviolacein. If the gene (vioC) for the conversion of
protodeoxyviolacein to deoxyviolacein, which is a purple pigment, is missing,
a metabolic shift takes place towards prodeoxyviolacein, which presents a
green color. (F) The pDrop plasmid harbours a partly duplicated, non-
functional mScarlet gene. During Drop recombination, the pDrop plasmid is cut
by the sgRNA TM, which is expressed on the pSwap plasmid and subsequently
recombined resulting in a functional mScarlet gene. Colonies appear red after
successful recombination.  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 4: Enhanced editing efficiency of green
colonies. (A) Shown is the editing efficiency of green and non-green colonies
for the reconstituted lacZ and araB genes. Experiments were carried out in
triplicates. 45 green and 45 non-green colonies were tested in total for both
lacZ and araB reconstitution. (B) Shown is the color distribution of colonies
in three different lacZ reconstitution experiments. (C) Shown is the editing
efficiency of green colonies of single and multiplex editing. Error bars
represent the standard error. Experiments were carried out in triplicates. For
dual editing (lacZ and araB), 45 colonies and for the quadruple edit (lacZ,
araB, xylA and dapA), 38 colonies were tested in total.  Figure 5: 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${\Delta}$del4)
and the edited strain (E. coli${\Delta}$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${\Delta}$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${\Delta}$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${\Delta}$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.  Figure
6: Integration of a 151kbp E. coli chromosomal region into the V. natriegens
chromosome. (A) Shown are agarose gels of PCRs from the transfer and
integration of a 151kbp chromosomal region from E. coli into the recJ locus of
V. natriegens. PCRs were done for the acceptor strain (V. natriegens WT) and
for the edited strain (V. natriegens recJ::MFDdel4). PCRs "1-6" result in the
amplification of evenly distributed fragments of the E. coli del4 region
within the V. natriegens chromosome. PCR "7" results in the amplification of a
4479bp fragment, if the recJ locus is unchanged. PCR "8" and "9" amplify the
transition of the del4 region to the adjacent chromosomal regions. (B) Shown
is the next-generation sequencing (NGS) coverage of the edited strain V.
natriegens recJ::MFDdel4 (light grey) compared to the reference V. natriegens
WT strain (dark grey). Both NGS reads were aligned against the edited
reference genome (recJ::MFDdel4). The sector of the del4 insertion as well as
the complete genome coverage (circle) are shown. Reads of the complete del4
insertion (between dashed lines) are present for the edited strain, while no
reads are present for the wild-type strain (red rectangle).  Figure 7:
Functionality of the E. coli lactose operons in V. natriegens. (A) Shown is a
picture of V. natriegens colonies, harbouring the E. coli DH5${\alpha}$ lac
operon, transformed with a plasmid containing the lacZ $\alpha$ fragment.
Cells were plated on LBv2 agar supplemented with IPTG and X-gal. (B) Shown is
the gene map of the V. natriegens strains tested for lactose metabolism. The
native recJ gene is replaced by the E. coli lac operon in V. natriegens lacEc.
This strain was further edited by the elimination of the lacEc operon
resulting in V. natriegens $\Delta$lacEc, which is also a $\Delta$recj knock
out. (C) Shown are the growth rates of V. natriegens strains and E. coli grown
in different media and carbon sources. For each sample, six independent
replicates were performed. Cells were grown in M9 minimal medium supplemented
with either 0.4% glucose, lactose and galactose as well as in LB medium using
a microplate reader. Error bars represent the standard error. Figure 8:
Enhanced editing efficiency of green colonies in V. natriegens. (A) Shown is
the editing efficiency of green and non-green colonies for the reconstituted
lacZ gene. Experiments were carried out in triplicates. In total, 43 green and
32 non-green colonies were tested. (B) Shown is the color distribution of
colonies in three different lacZ reconstitution experiments. Error bars
represent the standard error.  Figure S1: Colony and culture colors of the
multi-color selection system. (A) Shown is a picture of purple colonies
representing a successful pSwap assembly. (B) Shown is a picture of green
colonies after a successful editing event (SWAP). (C) Shown is a picture of
red colonies after a successful editing event (DROP). (D) Shown is a picture
of bacterial cultures, each with one of the 3 different colors (purple, green,
red) used for selection.  Figure S2: Iterative genome editing with CRISPR
SWAPnDROP. The use of the two pSwap plasmids (CmR/GmR) allows iterative genome
editing. Each harbouring either the I-SceI or I-CreI meganuclease genes and
the opposite recognition site I-CreI site or I-SceI site, respectively. During
the editing the meganuclease genes are removed leaving the pSwap plasmid only
with the opposite recognition site. Subsequent transformation of the other
pSwap plasmid leads to the elimination of the previous plasmid and allows
another round of editing. Figure S3: Mechanism of CRISPR SWAPnDROP
recombination for multiplex genome editing. CRISPR SWAPnDROP can also be used
for the simultaneous edit of two chromosomal regions. For this purpose, two
templates (HA-INS-HB) for recombination are assembled together and separated
by an additional Cas9 excision site between HB1 and HA2. T1 and T2 sgRNAs
targeting two different chromosomal loci are used for counter-selection.
Figure S4: Transfer and integration of a 151kbp chromosomal region via CRISPR
SWAPnDROP. To demonstrate the transfer of large chromosomal regions, an E.
coli MG1655 strain was first generated lacking a 167kbp chromosomal region
(del4) near the terminus of the E. coli chromosome (E. coli${\Delta}$del4). E.
coli MFDpir was then used to load the pSwap plasmid with the MFDdel4 region
(151kbp) using H1 and H2, which are equivalent to HA and HB in the E.
coli${\Delta}$del4 strain. The loaded pSwap’ was then conjugated into E.
coli${\Delta}$del4. For the reconstitution of the E. coli${\Delta}$del4
strain, H${\alpha}$ and H${\beta}$ were used to drop the 151kbp region into
the deleted del4 region. This resulted in the strain E.
coli${\Delta}$del4::MFDdel4, which lacks small regions between H${\alpha}$/HA
and HB/H${\beta}$.  Figure S5: 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.