scholarly journals CRISPR-Cas12a-Assisted Recombineering in Bacteria

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Mei-Yi Yan ◽  
Hai-Qin Yan ◽  
Gai-Xian Ren ◽  
Ju-Ping Zhao ◽  
Xiao-Peng Guo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mycobacterium Smegmatis ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Gene ◽  
Point Mutations ◽  
Content Type ◽  
Short Palindromic Repeat ◽  
New Type ◽  
System Point ◽  
Selection Of

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis. Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria. IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

scholarly journals Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

2015 ◽  
Vol 197 (6) ◽  
pp. 1135-1144 ◽  
Author(s):  
Jennifer F. Carr ◽  
Michael E. Danziger ◽  
Athena L. Huang ◽  
Albert E. Dahlberg ◽  
Steven T. Gregory
Keyword(s):  
Resistance Gene ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Thermus Thermophilus ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Biological Research ◽  
Lethal Mutant ◽  
Α Subunit ◽  
Content Type

ABSTRACTThermus thermophilusis an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele ofpheSencoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analogp-chlorophenylalanine. Insertion of the mutantpheSallele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination usingp-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of therrsBgene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of theT. thermophilusinsertion elements ISTth7and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on β-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome ofT. thermophilusand possibly other thermophiles as well.IMPORTANCEThermus thermophilusis an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into theT. thermophilusgenome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

scholarly journals History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology

2018 ◽  
Vol 200 (7) ◽  
Author(s):  
Yoshizumi Ishino ◽  
Mart Krupovic ◽  
Patrick Forterre
Keyword(s):  
Genome Editing ◽  
Functional Characterization ◽  
Halophilic Archaea ◽  
Repeated Sequence ◽  
Hyperthermophilic Archaea ◽  
Content Type ◽  
Origin And Evolution ◽  
History Of ◽  
Short Palindromic Repeat ◽  
Original Observation

ABSTRACTClustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in theEscherichia coligenome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence inE. colito genome editing in humans.

scholarly journals Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Kyunghun Min ◽  
Yuichi Ichikawa ◽  
Carol A. Woolford ◽  
Aaron P. Mitchell
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Drug Targets ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Content Type ◽  
Biological Features ◽  
Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also discussed.

scholarly journals Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
J. B. Buil ◽  
H. A. L. van der Lee ◽  
A. J. M. M. Rijs ◽  
J. Zoll ◽  
J. A. M. F. Hovestadt ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sensitivity And Specificity ◽  
Antifungal Susceptibility ◽  
Screening Method ◽  
Point Mutations ◽  
Azole Resistance ◽  
Minimal Growth ◽  
Content Type ◽  
Mean Sensitivity ◽  
Selection Of

ABSTRACT Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

scholarly journals A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mei-Yi Yan ◽  
Si-Shang Li ◽  
Xin-Yuan Ding ◽  
Xiao-Peng Guo ◽  
Qi Jin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genome Editing ◽  
Large Scale ◽  
Nonhomologous End Joining ◽  
Genetic Mutations ◽  
Repair Pathway ◽  
End Joining ◽  
Content Type ◽  
Nhej Repair ◽  
Short Palindromic Repeat

ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

scholarly journals Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Brian M. Luna ◽  
Amber Ulhaq ◽  
Jun Yan ◽  
Paul Pantapalangkoor ◽  
Travis B. Nielsen ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Acinetobacter Baumannii ◽  
Genetic Manipulation ◽  
Multidrug Resistant ◽  
Molecular Techniques ◽  
Drug Resistant ◽  
Selectable Markers ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Selection Of

ABSTRACT Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics. Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

scholarly journals Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System

2020 ◽  
Vol 8 (5) ◽  
pp. 754
Author(s):  
Youran Li ◽  
Hanrong Wang ◽  
Liang Zhang ◽  
Zhongyang Ding ◽  
Sha Xu ◽  
...  
Keyword(s):  
Success Rate ◽  
Genome Editing ◽  
Bacillus Licheniformis ◽  
Genetic Manipulation ◽  
Inducible Promoter ◽  
Bacterial Cells ◽  
Protospacer Adjacent Motif ◽  
Constitutive Overexpression ◽  
Short Palindromic Repeat ◽  
Overexpression System

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

scholarly journals Efficient Counterselection forMethylococcus capsulatus(Bath) by Using a MutatedpheSGene

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Masahito Ishikawa ◽  
Sho Yokoe ◽  
Souichiro Kato ◽  
Katsutoshi Hori
Keyword(s):  
Genetic Manipulation ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Efficient Production ◽  
Gram Negative Bacteria ◽  
Methylococcus Capsulatus ◽  
Α Subunit ◽  
Gram Negative ◽  
Content Type ◽  
Research Fields

ABSTRACTMethylococcus capsulatus(Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. ThesacBgene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However,sacBis not applicable to all Gram-negative bacteria, and its efficiency for the counterselection ofM. capsulatus(Bath) is low. Here, we report the construction of an alternative counterselectable marker,pheS*, by introduction of two point mutations (A306G and T252A) into thepheSgene fromM. capsulatus(Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboringpheS* on an expression plasmid showed sensitivity to 10 mMp-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability ofpheS* as a counterselectable marker inM. capsulatus(Bath). To validate the utility of thepheS* marker in counterselection, we attempted to obtain an unmarked mutant ofxoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using thesacBmarker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of thexoxFgene inM. capsulatus(Bath). The difference in counterselection efficiencies of the markers indicated thatpheS* is more suitable thansacBfor counterselection inM. capsulatus(Bath). This study provides a new genetic tool enabling efficient counterselection inM. capsulatus(Bath).IMPORTANCEMethanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such asEscherichia coli. Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection inM. capsulatus(Bath) by employingpheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants ofM. capsulatus(Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.

scholarly journals Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

2021 ◽  
Vol 7 (11) ◽  
Author(s):  
Juan Pablo Gomez-Escribano ◽  
Lis Algora Gallardo ◽  
Kenan A. J. Bozhüyük ◽  
Steven G. Kendrew ◽  
Benjamin D. Huckle ◽  
...  
Keyword(s):  
Genome Editing ◽  
Type Strain ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Sequence Data ◽  
Streptomyces Clavuligerus ◽  
Sequence Coverage ◽  
Content Type ◽  
Large Deletions ◽  
Low Levels

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

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