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.

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

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 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 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 PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0149762 ◽  
Author(s):  
Yilan Liu ◽  
Maohua Yang ◽  
Jinjin Chen ◽  
Daojiang Yan ◽  
Wanwan Cheng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

2021 ◽  
Vol 53 (5) ◽  
pp. 620-627
Author(s):  
Qi Li ◽  
Bingbing Sun ◽  
Jun Chen ◽  
Yiwen Zhang ◽  
Yu Jiang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency ◽  
Specific Sequence ◽  
E Coli ◽  
Strain Bl21 ◽  
Sacb Gene ◽  
Ccdb Gene ◽  
K 12 ◽  
Plasmid Elimination

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.

scholarly journals Characterizing the portability of RecT-mediated oligonucleotide recombination

2020 ◽  
Author(s):  
Gabriel T. Filsinger ◽  
Timothy M. Wannier ◽  
Felix B. Pedersen ◽  
Isaac D. Lutz ◽  
Julie Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
Mycobacterium Smegmatis ◽  
High Efficiency ◽  
Caulobacter Crescentus ◽  
Bacterial Genome ◽  
Lactobacillus Rhamnosus ◽  
Dna Binding Protein ◽  
Fundamental Research ◽  
Terminal Amino

AbstractBacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the hostlimited functionality of RecTs. We find that these proteins specifically recognize the 7 C-terminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through sequential nucleotide conversions.

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

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
Cloning Strategy

scholarly journals Multiplex genome editing by natural transformation (MuGENT) for synthetic biology inVibrio natriegens

2017 ◽  
Author(s):  
Triana N. Dalia ◽  
Chelsea A. Hayes ◽  
Sergey Stolyar ◽  
Christopher J. Marx ◽  
James B. McKinlay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Genome Editing ◽  
High Efficiency ◽  
Natural Transformation ◽  
Value Added ◽  
Industrial Applications ◽  
Proof Of Concept ◽  
Natural Competence ◽  
Low Efficiency

ABSTRACTVibrio natriegenshas recently emerged as an alternative toEscherichia colifor molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence inV. natriegensand describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented timescales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) inV. natriegensby targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ~100 times more PHB than the parent isolate and ~3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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