scholarly journals Response to Comment on “RNA-guided DNA insertion with CRISPR-associated transposases”

Science ◽  
2020 ◽  
Vol 368 (6495) ◽  
pp. eabb2920 ◽  
Author(s):  
Jonathan Strecker ◽  
Alim Ladha ◽  
Kira S. Makarova ◽  
Eugene V. Koonin ◽  
Feng Zhang
Keyword(s):  
Escherichia Coli ◽  
Donor Plasmid ◽  
Dna Insertion ◽  
Simple Integration

Rice et al. suggest that the CRISPR-associated transposase ShCAST system could lead to additional insertion products beyond simple integration of the donor. We clarify the outcomes of ShCAST-mediated insertions in Escherichia coli, which consist of both simple insertions and integration of the donor plasmid. This latter outcome can be avoided by use of a 5′ nicked DNA donor.

scholarly journals UV Light Induces IS10 Transposition in Escherichia coli

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1173-1181
Author(s):  
Zehava Eichenbaum ◽  
Zvi Livneh
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Target Gene ◽  
Uv Light ◽  
Insertion Site ◽  
Donor Plasmid ◽  
Donor And Acceptor ◽  
Low Copy Number ◽  
Uv Induction ◽  
Target Plasmid

Abstract A new mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid was used to investigate the effect of UV light on intermolecular transposition of IS10. Inactivation of the target gene by IS10 insertion was detected by the expression of the tet gene from the phage 434 PR promoter, followed by Southern blot analysis of plasmids isolated from TetR colonies. UV irradiation of cells harboring the target plasmid and a donor plasmid carrying an IS10 element led to an increase of up to 28-fold in IS10 transposition. Each UV-induced transposition of IS10 was accompanied by fusion of the donor and acceptor plasmid into a cointegrate structure, due to coupled homologous recombination at the insertion site, similar to the situation in spontaneous IS10 transposition. UV radiation also induced transposition of IS10 from the chromosome to the target plasmid, leading almost exclusively to the integration of the target plasmid into the chromosome. UV induction of IS10 transposition did not depend on the umuC and uvrA gene product, but it was not observed in lexA3 and ΔrecA strains, indicating that the SOS stress response is involved in regulating UV-induced transposition. IS10 transposition, known to increase the fitness of Escherichia coli, may have been recruited under the SOS response to assist in increasing cell survival under hostile environmental conditions. To our knowledge, this is the first report on the induction of transposition by a DNA-damaging agent and the SOS stress response in bacteria.

Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

1999 ◽  
Vol 181 (22) ◽  
pp. 7143-7148 ◽  
Author(s):  
F. Martinez-Morales ◽  
A. C. Borges ◽  
A. Martinez ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Helper Plasmid ◽  
Flp Recombinase ◽  
E Coli ◽  
Homologous Fragment ◽  
Genes Encoding ◽  
Dna Insertion ◽  
K 12 ◽  
Multiple Cloning Sites

ABSTRACT A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

scholarly journals High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

2014 ◽  
Vol 80 (13) ◽  
pp. 3826-3834 ◽  
Author(s):  
Junjie Yang ◽  
Bingbing Sun ◽  
He Huang ◽  
Yu Jiang ◽  
Liuyang Diao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Modification ◽  
Target Gene ◽  
Donor Plasmid ◽  
Content Type ◽  
Recognition Sites ◽  
Genetic Modifications ◽  
Pcr Targeting ◽  
Λ Red ◽  
Red Recombination

ABSTRACTGenetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of theEscherichia colichromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used forcadAknockout,gdhAknock-in, seamless deletion ofpepD, site-directed mutagenesis of the essentialmetKgene, and replacement ofmetKwith theRickettsiaS-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

scholarly journals Distribution of DNA insertion element IS5 in natural isolates of Escherichia coli.

1984 ◽  
Vol 81 (14) ◽  
pp. 4500-4504 ◽  
Author(s):  
L. Green ◽  
R. D. Miller ◽  
D. E. Dykhuizen ◽  
D. L. Hartl
Keyword(s):  
Escherichia Coli ◽  
Insertion Element ◽  
Natural Isolates ◽  
Dna Insertion

scholarly journals tDNA locus polymorphism and ecto-chromosomal DNA insertion hot-spots are related to the phylogenetic group of Escherichia coli strains

Microbiology ◽  
2007 ◽  
Vol 153 (3) ◽  
pp. 826-837 ◽  
Author(s):  
Pierre Germon ◽  
David Roche ◽  
Sandrine Melo ◽  
Sandrine Mignon-Grasteau ◽  
Ulrich Dobrindt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hot Spots ◽  
Phylogenetic Group ◽  
Chromosomal Dna ◽  
Dna Insertion

scholarly journals JOINT DISTRIBUTION OF INSERTION ELEMENTS IS4 AND IS5 IN NATURAL ISOLATES OF ESCHERICHIA COLI

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 219-231
Author(s):  
Daniel E Dykhuizen ◽  
Stanley A Sawyer ◽  
Louis Green ◽  
Raymond D Miller ◽  
Daniel L Hartl
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Significant Negative Correlation ◽  
Single Copy ◽  
Common Mechanism ◽  
Reference Collection ◽  
Insertion Elements ◽  
Natural Isolates ◽  
Dna Insertion ◽  
Flanking Sequences

ABSTRACT A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 ± 0.8; the comparable figure for IS5 was 3.7 ± 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements.— The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.

scholarly journals Comment on “RNA-guided DNA insertion with CRISPR-associated transposases”

Science ◽  
2020 ◽  
Vol 368 (6495) ◽  
pp. eabb2022 ◽  
Author(s):  
Phoebe A. Rice ◽  
Nancy L. Craig ◽  
Fred Dyda
Keyword(s):  
Research Articles ◽  
Great Promise ◽  
Donor Plasmid ◽  
Dna Insertion

Strecker et al. (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.

scholarly journals RNA-guided DNA insertion with CRISPR-associated transposases

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. 48-53 ◽  
Author(s):  
Jonathan Strecker ◽  
Alim Ladha ◽  
Zachary Gardner ◽  
Jonathan L. Schmid-Burgk ◽  
Kira S. Makarova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acids ◽  
Positive Selection ◽  
Host Cell ◽  
Functional Diversity ◽  
Base Pairs ◽  
Dna Transposition ◽  
Cell Repair ◽  
Dna Insertion ◽  
Type V

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

scholarly journals Differential Spectrum of Mutations That Activate the Escherichia coli bgl Operon in an rpoS Genetic Background

2002 ◽  
Vol 184 (14) ◽  
pp. 4033-4038 ◽  
Author(s):  
Sudha Moorthy ◽  
S. Mahadevan
Keyword(s):  
Escherichia Coli ◽  
Natural Populations ◽  
Structural Elements ◽  
Wild Type ◽  
Genetic Changes ◽  
E Coli ◽  
Activating Mutations ◽  
Major Class ◽  
Insertion Elements ◽  
Dna Insertion

ABSTRACT The bgl promoter is silent in wild-type Escherichia coli under standard laboratory conditions, and as a result, cells exhibit a β-glucoside-negative (Bgl−) phenotype. Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS. Mutations that confer a Bgl+ phenotype arise spontaneously at a detectable frequency. Transposition of DNA insertion elements within the regulatory locus, bglR, constitutes the major class of activating mutations identified in laboratory cultures. The rpoS-encoded σS, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions. In an attempt to see if the rpoS status of the cell influences the nature of the mutations that activate the bgl promoter, we analyzed spontaneously arising Bgl+ mutants in rpoS+ and rpoS genetic backgrounds. We show that the spectrum of activating mutations in rpoS cells is different from that in rpoS+ cells. Unlike rpoS+ cells, where insertions in bglR are the predominant activating mutations, mutations in hns make up the majority in rpoS cells. The physiological significance of these differences is discussed in the context of survival of natural populations of E. coli.

scholarly journals Diversity of Tn4001 Transposition Products: the Flanking IS256 Elements Can Form Tandem Dimers and IS Circles

2002 ◽  
Vol 184 (2) ◽  
pp. 433-443 ◽  
Author(s):  
M. Prudhomme ◽  
C. Turlan ◽  
J.-P. Claverys ◽  
M. Chandler
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Sequence Analysis ◽  
Higher Order ◽  
The Other ◽  
Insertion Sequences ◽  
Donor Plasmid ◽  
Sequence Alignments ◽  
Vector Plasmid ◽  
Junction Sequence

ABSTRACT We show that both flanking IS256 elements carried by transposon Tn4001 are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256 belonging to one Tn4001 copy attacks an IS256 end carried by the second Tn4001 copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256 ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis in Escherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256 as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001 is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such as Streptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.

Sign in / Sign up

Export Citation Format

Share Document