scholarly journals Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

2018 ◽  
Author(s):  
Wenqiang Li ◽  
Shuntang Li ◽  
Jie Qiao ◽  
Fei Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
General Strategy ◽  
Dna Assembly ◽  
E Coli ◽  
Assembly Method ◽  
Direct Preparation

AbstractCRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

Expression, purification and properties of multidrug efflux proteins

2000 ◽  
Vol 28 (4) ◽  
pp. 513-517 ◽  
Author(s):  
P.J. F. Henderson ◽  
C. K. Hoyle ◽  
A. Ward
Keyword(s):  
Fluorescent Protein ◽  
Affinity Column ◽  
General Strategy ◽  
X Rays ◽  
Multidrug Efflux ◽  
Nmr Studies ◽  
E Coli ◽  
Efflux Proteins

A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H+ antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1–25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni2+-nitrilo-triacetate-affinity column chromography, yielding 5–25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of α-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with 13C or 15N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

scholarly journals Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court
Keyword(s):  
Dna Assembly ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Engineering Technology ◽  
E Coli ◽  
Linear Dna ◽  
Recombination System ◽  
Genetic Engineering Technology

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

scholarly journals Genome-wide CRISPR-Cas9 screen in E. coli identifies design rules for efficient targeting

2018 ◽  
Author(s):  
Belen Gutierrez ◽  
Jérôme Wong Ng ◽  
Lun Cui ◽  
Christophe Becavin ◽  
David Bikard
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Mixed Population ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
A Genome

AbstractThe main outcome of efficient CRISPR-Cas9 cleavage in the chromosome of bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population of bacteria, which can be achieved both in vitro, e.g. to select mutants, or in vivo as an antimicrobial strategy. The efficiency with which Cas9 kills bacteria has been observed to be quite variable depending on the specific target sequence, but little is known about the sequence determinants and mechanisms involved. Here we performed a genome-wide screen of Cas9 cleavage in the chromosome of E. coli to determine the efficiency with which each guide RNA kills the cell. Surprisingly we observed a large-scale pattern where guides targeting some regions of the chromosome are more rapidly depleted than others. Unexpectedly, this pattern arises from the influence of degrading specific chromosomal regions on the copy number of the plasmid carrying the guide RNA library. After taking this effect into account, it is possible to train a neural network to predict Cas9 efficiency based on the target sequence. We show that our model learns different features than previous models trained on Eukaryotic CRISPR-Cas9 knockout libraries. Our results highlight the need for specific models to design efficient CRISPR-Cas9 tools in bacteria.

scholarly journals Bacterial Artificial Chromosome Mutagenesis Using Recombineering

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Kumaran Narayanan ◽  
Qingwen Chen
Keyword(s):  
Gene Expression ◽  
Bacterial Artificial Chromosome ◽  
Restriction Enzymes ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
E Coli ◽  
Functional Studies ◽  
Large Size

Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development ofin vivohomologous recombination strategies based on recombineering inE. colihas helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACsin vitroandin vivo.

scholarly journals Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Satoru Iwata ◽  
Hitomi Nakadai ◽  
Daisuke Fukushi ◽  
Mami Jose ◽  
Miki Nagahara ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genetically Engineered ◽  
Guide Rna ◽  
Chromosomal Inversions ◽  
Genetically Engineered Mice ◽  
Cas9 Protein ◽  
Mouse Zygotes

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.

scholarly journals In vitro turnover numbers do not reflect in vivo activities of yeast enzymes

2021 ◽  
Vol 118 (32) ◽  
pp. e2108391118
Author(s):  
Yu Chen ◽  
Jens Nielsen
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Metabolic Model ◽  
Proteomics Data ◽  
Weak Correlation ◽  
E Coli ◽  
Activity Of Enzymes

Turnover numbers (kcat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (kapp values) in bacteria, a large-scale estimation of kapp in eukaryotes is lacking. Here, we estimated kapp of the yeast Saccharomyces cerevisiae under diverse conditions. By comparing the maximum kapp across conditions with in vitro kcat we found a weak correlation in log scale of R2 = 0.28, which is lower than for Escherichia coli (R2 = 0.62). The weak correlation is caused by the fact that many in vitro kcat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to R2 = 0.41 but still not as good as for E. coli, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our kapp dataset we observed better performance than the default model with in vitro kcat in predicting proteomics data, demonstrating the strength of using the dataset generated here.

scholarly journals Screening for functional circular RNAs using the CRISPR-Cas13 system

2020 ◽  
Author(s):  
Siqi Li ◽  
Xiang Li ◽  
Wei Xue ◽  
Lin Zhang ◽  
Shi-Meng Cao ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Functional Study ◽  
Circular Rnas ◽  
Rna Targeting ◽  
Scale Levels ◽  
A Cell ◽  
Cell Type Specific

SummaryCircular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood due to inadequate methods, such as RNAi and genome engineering, in distinguishing overlapped exons in circRNAs from those in linear cognate mRNAs1,2. Here we report that the programable RNA-guided, RNA-targeting CRISPR-Cas13, RfxCas13d, effectively and specifically discriminates circRNAs from mRNAs, using guide (g)RNAs targeting sequences spanning the back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation in vitro and in vivo by preventing FAM120A mRNA from binding the translation inhibitor IGF2BP2 for efficient translation. Application of RfxCas13d/BSJ-gRNA screening has also uncovered circMan1a2 with regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

scholarly journals Direct purification of CRISPR/Cas ribonucleoprotein from E. coli in a single step

2018 ◽  
Author(s):  
Siyu Lin ◽  
Jie Qiao ◽  
Lixin Ma ◽  
Yi Liu
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Cost Effective ◽  
Nuclease Activity ◽  
Single Step ◽  
Simple Method ◽  
E Coli ◽  
Purification System

AbstractCRISPR/Cas ribonucleoprotein (RNP) complexes have been recently used as promising biological tools with plenty of applications, however, there are by far no efficient methods to prepare them at large scale and low cost. Here, we present a simple method to directly produce and purify Cas RNP, including the widely used Cas9 and Cas12a nuclease, from E.coli in a single step using an ultra-high-affinity CL7/Im7 purification system. The prepared Cas RNP shows high stability, solid nuclease activity in vitro, and profound genome editing efficiency in vivo. Our method is convenient, cost-effective, and applicable to prepare other CRISPR associated nucleases.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Sign in / Sign up

Export Citation Format

Share Document