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.

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

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 An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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

scholarly journals Orthogonal CRISPR-Cas genome editing and efficient inhibition with anti-CRISPRs in zebrafish embryos

2020 ◽  
Author(s):  
Paige R. Takasugi ◽  
Evan P. Drage ◽  
Sahar N. Kanishka ◽  
Marissa A. Higbee ◽  
James A. Gagnon
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
High Efficiency ◽  
Bacterial Species ◽  
Zebrafish Embryos ◽  
Type Ii ◽  
Guide Rnas ◽  
Highly Effective ◽  
Orthogonal Systems ◽  
Spatiotemporal Control

AbstractThe CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system limits options for multiplexed editing. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpCas9), Streptococcus aureus (SaCas9), and Lachnospiraceae bacterium (LbCas12a, previously known as LbCpf1) are highly effective, orthogonal systems capable of operating simultaneously in zebrafish. We also demonstrate that type II Acrs are effective inhibitors of SpCas9 in zebrafish. These results indicate that at least three orthogonal CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing in zebrafish.

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.

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 Cas9-mediated genome editing in the methanogenic archaeonMethanosarcina acetivorans

2017 ◽  
Vol 114 (11) ◽  
pp. 2976-2981 ◽  
Author(s):  
Dipti D. Nayak ◽  
William W. Metcalf
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Genetic System ◽  
Nonhomologous End Joining ◽  
Methanosarcina Acetivorans ◽  
End Joining ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Insertion And Deletion

Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeonMethanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon,Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.

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.

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