scholarly journals Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

2020 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombination Event ◽  
Plasmid Stability ◽  
Direct Repeats ◽  
Replication Slippage ◽  
High Frequencies ◽  
Plasmid Construction ◽  
Guide Rnas ◽  
Cloning Strategy ◽  
Chromosome Fragments

Abstract Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

scholarly journals Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

2020 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombination Event ◽  
Plasmid Stability ◽  
Direct Repeats ◽  
Replication Slippage ◽  
High Frequencies ◽  
Plasmid Construction ◽  
Guide Rnas ◽  
Cloning Strategy ◽  
Chromosome Fragments

Abstract Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli.Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

2019 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency ◽  
Direct Repeats ◽  
Replication Slippage ◽  
High Frequencies ◽  
Plasmid Instability ◽  
Plasmid Construction ◽  
Cloning Strategy ◽  
Chromosome Fragments

SummaryA growing number of CRISPR-Cas9 associated applications require co-expression of two distinct gRNAs. However, coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid instability, due to the presence of direct repeats (DRs). In this study, deletion between DRs occurred with high frequencies during plasmid construction and duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). Using RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that relied on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

scholarly journals Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

2019 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency ◽  
Replication Slippage ◽  
High Frequencies ◽  
Guide Rnas ◽  
Large Deletions ◽  
Plasmid Recombination ◽  
Cloning Strategy ◽  
Chromosome Fragments

Abstract BackgroundThe CRISPR-Cas9 system is a powerful tool for genome editing in various organisms. Several of its applications, including the generation of large deletions, require co-expression of two distinct guide RNAs (gRNAs). However, the instability of paired-gRNA plasmids prevents these applications from being scalable in Escherichia coli. Coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid recombination, due to the presence of direct repeats (DRs). ResultsIn this study, plasmid deletion between DRs occurred with high frequencies during plasmid construction and subsequent duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). ConclusionsUsing RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that rely on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

scholarly journals Deletion Formation Between the Two Salmonella typhimurium Flagellin Genes Encoded on the Mini F Plasmid: Escherichia coli ssb Alleles Enhance Deletion Rates and Change Hot-Spot Preference for Deletion Endpoints

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 563-572 ◽  
Author(s):  
Takafumi Mukaihara ◽  
Masatoshi Enomoto
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Hot Spots ◽  
Hot Spot ◽  
Direct Repeats ◽  
Replication Slippage ◽  
Detection Systems ◽  
Deletion Formation ◽  
Homologous Sequences ◽  
Deletion Detection

Deletion formation between the 5′-mostly homologous sequences and between the 3′-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5′ sequences, but not between the 3′ sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the ΔrecA allele. Rates of the deletion between the 3′ sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA+ background and 88% in ΔrecA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background >50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3′ sequences. The deletion formation between the homeologous sequences is RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.

scholarly journals Creation of a Chloroplast Microsatellite Reporter for Detection of Replication Slippage in Chlamydomonas reinhardtii

2008 ◽  
Vol 7 (4) ◽  
pp. 639-646 ◽  
Author(s):  
Monica GuhaMajumdar ◽  
Ethan Dawson-Baglien ◽  
Barbara B. Sears
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Forensic Analysis ◽  
Direct Repeats ◽  
Reporter Construct ◽  
Chloroplast Microsatellite ◽  
Replication Slippage ◽  
Reading Frame ◽  
Viable Cells ◽  
Short Tandem

ABSTRACT Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 × 10−5. Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule.

Fermentation strategy to enhance plasmid stability during the cultivation of Escherichia coli for the production of recombinant levansucrase

1998 ◽  
Vol 86 (4) ◽  
pp. 391-394 ◽  
Author(s):  
Chul Ho Kim ◽  
Jang Young Lee ◽  
Min Gon Kim ◽  
Ki Bang Song ◽  
Jeong Woo Seo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Fermentation Strategy

scholarly journals Antibiotic breakdown by susceptible bacteria enhances the establishment of β-lactam resistant mutants

2021 ◽  
Author(s):  
Manja Saebelfeld ◽  
Suman G Das ◽  
Jorn Brink ◽  
Arno Hagenbeek ◽  
Joachim Krug ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Theoretical Model ◽  
Enzymatic Activity ◽  
Social Interactions ◽  
Single Cells ◽  
Antibiotic Concentration ◽  
High Frequencies ◽  
Establishment Probability ◽  
Resistant Mutants

For a better understanding of the evolution of antibiotic resistance, it is imperative to study the factors that determine the initial establishment of mutant resistance alleles. In addition to the antibiotic concentration, the establishment of resistance alleles may be affected by interactions with the surrounding susceptible cells from which they derive, for instance via the release of nutrients or removal of the antibiotic. Here, we investigate the effects of social interactions with surrounding susceptible cells on the establishment of Escherichia coli mutants with increasing β-lactamase activity (i.e. the capacity to hydrolyze β-lactam antibiotics) from single cells under the exposure of the antibiotic cefotaxime on agar plates. We find that mutant establishment probability is increased in the presence of susceptible cells due to the active breakdown of the antibiotic, but the rate of breakdown by the susceptible strain is much higher than expected based on its low enzymatic activity. A detailed theoretical model suggests that this observation can be explained by cell filamentation causing delayed lysis. While susceptible cells may hamper the spread of higher-resistant β-lactamase mutants at relatively high frequencies, our findings show that they could promote establishment during their emergence.

scholarly journals The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

2020 ◽  
Vol 21 (2) ◽  
pp. 576 ◽  
Author(s):  
Luciana Gomes ◽  
Gabriel Monteiro ◽  
Filipe Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
Chemical Induction ◽  
T7 Promoter ◽  
Iptg Induction ◽  
The Impact

This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

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