CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit for E. coli

AbstractIn most prokaryotes, missing and poorly active non-homologous end joining (NHEJ) DNA repair pathways heavily restrict the direct application of CRISPR-Cas for DNA double-strand break (DSB)-based genome engineering without providing editing templates. CRISPR base editors, on the other hand, can be directly used for genome engineering in a number of bacteria, including E. coli, showing advantages over CRISPR-Cas9, since they do not require DSBs. However, as the current CRISPR base editors can only engineer DNA by A to G or C to T/G/A substitutions, they are incapable of mediating deletions, insertions, and combinations of deletions, insertions and substitutions. To address these challenges, we developed a Cas9 nickase (Cas9n)-reverse transcriptase (Moloney Murine Leukemia Virus, M-MLV) mediated, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes, termed CRISPR-nRAGE (CRISPR-Cas9n Reverse transcriptase Assisted Genome Engineering) system. CRISPR-nRAGE can be used to introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli. Notably, small sized-deletion shows better editing efficiency compared to other kinds of DNA engineering. CRISPR-nRAGE has been used to delete and insert DNA fragments up to 97 bp and 33 bp, respectively. Efficiencies, however, drop sharply with the increase of the fragment size. It is not only a useful addition to the genome engineering arsenal for E. coli, but also may be the basis for the development of similar toolkits for other organisms.

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A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

Nature Communications ◽

10.1038/s41467-021-25541-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yaojun Tong ◽

Tue S. Jørgensen ◽

Christopher M. Whitford ◽

Tilmann Weber ◽

Sang Yup Lee

Keyword(s):

Genome Engineering ◽

Genetic Manipulation ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rna ◽

E Coli ◽

Potential Basis ◽

Low Efficiency ◽

Nucleotide Resolution

AbstractCRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

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Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System

Biomolecules ◽

10.3390/biom8040137 ◽

2018 ◽

Vol 8 (4) ◽

pp. 137 ◽

Cited By ~ 3

Author(s):

Ying-Jie Yang ◽

Raghvendra Singh ◽

Xin Lan ◽

Cheng-Sheng Zhang ◽

Yue-Zhong Li ◽

...

Keyword(s):

Genome Engineering ◽

Genetic Manipulation ◽

Myxococcus Xanthus ◽

Gene Clusters ◽

Inducible Promoter ◽

Engineering System ◽

Suicide Vector ◽

Recombination System ◽

Gene Polymerase Chain Reaction ◽

Deletion Frequency

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.

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Purification and properties of Rauscher leukemia virus DNA polymerase and selective inhibition of mammalian viral reverse transcriptase by inorganic phosphate.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32792-8 ◽

1977 ◽

Vol 252 (1) ◽

pp. 11-19

Author(s):

M J Modak ◽

S L Marcus

Keyword(s):

Reverse Transcriptase ◽

Dna Polymerase ◽

Inorganic Phosphate ◽

Leukemia Virus ◽

Selective Inhibition ◽

Purification And Properties ◽

Rauscher Leukemia ◽

Rauscher Leukemia Virus

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Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Biotechnology for Biofuels ◽

10.1186/s13068-021-01883-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Anmoldeep Randhawa ◽

Nandita Pasari ◽

Tulika Sinha ◽

Mayank Gupta ◽

Anju M. Nair ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Engineering ◽

Efflux Pump ◽

Genetic Manipulation ◽

Function Analysis ◽

Drug Tolerance ◽

Growth Media ◽

Penicillium Funiculosum ◽

Cellobiohydrolase I ◽

Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae

Wellcome Open Research ◽

10.12688/wellcomeopenres.14642.1 ◽

2018 ◽

Vol 3 ◽

pp. 72

Author(s):

Peter W Daniels ◽

Anuradha Mukherjee ◽

Alastair SH Goldman ◽

Bin Hu

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Manipulation ◽

Strand Break ◽

Integration Strategy ◽

System A ◽

Target Dna ◽

Biosynthesis Gene ◽

Dna Fragment ◽

Integrative Vectors ◽

Yeast Genomes

Integrating a desired DNA sequence into yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans.

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Constructing a DNAzyme Delivery and Expression System for Escherichia coli: A Prototype for Phage Therapy

European Journal of Biology and Biotechnology ◽

10.24018/ejbio.2021.2.2.158 ◽

2021 ◽

Vol 2 (2) ◽

pp. 19-25

Author(s):

Hugo V. C. Oliveira ◽

Spartaco Astolfi-Filho ◽

Edmar V. Andrade

Keyword(s):

Escherichia Coli ◽

Protein Delivery ◽

Phage Therapy ◽

Leukemia Virus ◽

Expression System ◽

Constitutive Expression ◽

Primary Component ◽

Morphological Pattern ◽

Filamentous Form ◽

E Coli

Antisense oligonucleotides exhibit high potential for use as therapeutic agents. '10-23' DNAzymes are antisense molecules with a high chemical stability and catalytic efficiency. In the present study, we developed a phagemid containing a DNAzyme expression system regulated by two promoters. One of these promoters, pA1, promotes constitutive expression of Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT). The other promoter, plac, regulates transcription of the RNA substrate from which MoMuLV-RT produces the DNAzyme by reverse transcription. The ftsZ DNAzyme was used to validate this expression system in the phagemid, named pDESCP. ftsZ DNAzyme expression altered the morphological pattern of Escherichia coli from a bacillary to filamentous form. In E. coli FtsZ is the primary component of the cell division apparatus, forming a structure known as Z-ring, which is the place of division. It is suggested that the DNAzyme ftsZ is decreasing the translation of this protein. Delivery of pDESCP into F+ strain of E. coli cells, using VCSM13, and the possible insertion of other DNAzymes into the cassette makes this phagemid an important prototype for phage therapy.

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