scholarly journals NT-CRISPR: Combining natural transformation and CRISPR/Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens

2021 ◽  
Author(s):  
Daniel Stukenberg ◽  
Josef Hoff ◽  
Anna Faber ◽  
Anke Becker
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Natural Transformation ◽  
Single Base ◽  
Highly Efficient ◽  
Target Spectrum ◽  
A Genome ◽  
Wide Range ◽  
Resistance Markers ◽  
Type Sequence

The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for a wide range of projects. To fully harness the potential of this fascinating bacterium, convenient and highly efficient genome editing methods are indispensable to create novel strains, tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This process, called natural transformation, was tamed for genome editing. It displays a high efficiency and is able to mediate uptake of multiple DNA fragments, thereby allowing multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR/Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR/Cas9, targeting the wild type sequence, leading to death of non-edited cells. Through highly efficient cell killing with efficiencies of up to 99.999 %, integration of antibiotic resistance markers became dispensable and thus enabled scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100 % and further demonstrated its applicability for the simultaneous deletion of multiple chromosomal regions. Lastly, we demonstrated that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby massively broadens the target spectrum.

scholarly journals A general approach to high-efficiency perovskite solar cells by any antisolvent

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alexander D. Taylor ◽  
Qing Sun ◽  
Katelyn P. Goetz ◽  
Qingzhi An ◽  
Tim Schramm ◽  
...  
Keyword(s):  
Solar Cells ◽  
High Efficiency ◽  
Perovskite Solar Cells ◽  
Precursor Solution ◽  
Microstructural Characterization ◽  
Application Rate ◽  
Perovskite Layer ◽  
Highly Efficient ◽  
Application Procedure ◽  
Wide Range

AbstractDeposition of perovskite films by antisolvent engineering is a highly common method employed in perovskite photovoltaics research. Herein, we report on a general method that allows for the fabrication of highly efficient perovskite solar cells by any antisolvent via manipulation of the antisolvent application rate. Through detailed structural, compositional, and microstructural characterization of perovskite layers fabricated by 14 different antisolvents, we identify two key factors that influence the quality of the perovskite layer: the solubility of the organic precursors in the antisolvent and its miscibility with the host solvent(s) of the perovskite precursor solution, which combine to produce rate-dependent behavior during the antisolvent application step. Leveraging this, we produce devices with power conversion efficiencies (PCEs) that exceed 21% using a wide range of antisolvents. Moreover, we demonstrate that employing the optimal antisolvent application procedure allows for highly efficient solar cells to be fabricated from a broad range of precursor stoichiometries.

scholarly journals Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

2020 ◽  
Author(s):  
Rabinowitz Roy ◽  
Abadi Shiran ◽  
Almog Shiri ◽  
Offen Daniel
Keyword(s):  
Genome Editing ◽  
Point Mutations ◽  
Nucleotide Variation ◽  
Single Nucleotide Variation ◽  
Computational Tool ◽  
Single Nucleotide ◽  
Base Editing ◽  
A Genome ◽  
Wide Range ◽  
Reference Protein

ABSTRACTBase editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

scholarly journals Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sachiko Okamoto ◽  
Yasunori Amaishi ◽  
Izumi Maki ◽  
Tatsuji Enoki ◽  
Junichi Mineno
Keyword(s):  
Genome Editing ◽  
Single Base ◽  
Highly Efficient ◽  
Base Substitutions

scholarly journals Highly efficient genome editing of human hematopoietic stem cells via a nano-silicon-blade delivery approach

2017 ◽  
Vol 9 (6) ◽  
pp. 548-554 ◽  
Author(s):  
Yuan Ma ◽  
Xin Han ◽  
Oscar Quintana Bustamante ◽  
Ricardo Bessa de Castro ◽  
Kai Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Hematopoietic Stem ◽  
Highly Efficient ◽  
Nano Silicon ◽  
Delivery Approach

We provided a nano-blade chip for HSCs specific delivery with the properties of rapid, high efficiency and harmless.

scholarly journals Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

2018 ◽  
Vol 115 (31) ◽  
pp. E7379-E7388 ◽  
Author(s):  
Laura J. Smith ◽  
Jason Wright ◽  
Gabriella Clark ◽  
Taihra Ul-Hasan ◽  
Xiangyang Jin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Homologous Recombination ◽  
Genome Editing ◽  
High Efficiency ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
A Genome ◽  
Component Approach ◽  
Il2rg Gene

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

scholarly journals TMEPAI genome editing in triple negative breast cancer cells

2017 ◽  
Vol 26 (1) ◽  
pp. 14-8 ◽  
Author(s):  
Bantari W.K. Wardhani ◽  
Meidi U. Puteri ◽  
Yukihide Watanabe ◽  
Melva Louisa ◽  
Rianto Setiabudy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Genome Editing ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
High Efficiency ◽  
Knock Out ◽  
Tnbc Cell ◽  
A Genome ◽  
Tnbc Cell Line

Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.

scholarly journals hei-tag: a highly efficient tag to boost targeted genome editing

2021 ◽  
Author(s):  
Thomas Thumberger ◽  
Tinatini Tavhelidse ◽  
Jose Arturo Gutierrez-Triana ◽  
Rebekka Medert ◽  
Alex Cornean ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Genome Editing ◽  
Nuclear Localization ◽  
High Efficiency ◽  
Basic Research ◽  
Model Systems ◽  
Nuclear Localization Signals ◽  
Highly Efficient ◽  
Cas9 Protein ◽  
Steric Hinderance

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. To reach its target, the bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion of nuclear localization signals (NLSs) to the Cas9 protein. Additional domains such as FLAG- or myc-tags are added for immediate detection or straight-forward purification. To avoid steric hinderance impacting on activity, amino acid linkers are employed connecting Cas9 and additional domains. We present the 'hei-tag (high efficiency-tag)', boosting the activity of the wide variety of CRISPR/Cas genome editing tools. The addition of the hei-tag to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency in model systems ranging from fish to mammals, including tissue culture applications. This allows to instantly upgrade existing and potentially highly adapted systems as well as establish novel highly efficient tools.

scholarly journals Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

2020 ◽  
Vol 48 (W1) ◽  
pp. W340-W347 ◽  
Author(s):  
Roy Rabinowitz ◽  
Shiran Abadi ◽  
Shiri Almog ◽  
Daniel Offen
Keyword(s):  
Binding Site ◽  
Genome Editing ◽  
Point Mutation ◽  
Point Mutations ◽  
Computational Tool ◽  
Coding Sequences ◽  
Coding Sequence ◽  
Base Editing ◽  
A Genome ◽  
Wide Range

Abstract Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A deaminase enzyme is fused to a deactivated Cas and enables transition conversions. The diversified repertoire of base editors provides a wide range of base editing possibilities. However, existing base editors cannot induce transversion substitutions and activate only within a specified region relative to the binding site, thus, they cannot precisely correct every point mutation. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a point mutation. When a precise correction is impossible, BE-FF aims to mutate bystander nucleotides in order to induce synonymous corrections that will correct the coding sequence. To measure BE-FF practicality, we analysed a database of human pathogenic point mutations. Out of the transition mutations, 60.9% coding sequences could be corrected. Notably, 19.4% of the feasible corrections were not achieved by precise corrections but only by synonymous corrections. Moreover, 298 cases of transversion-derived pathogenic mutations were detected to be potentially repairable by base editing via synonymous corrections, although base editing is considered impractical for such mutations.

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.

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