scholarly journals Gene editing enables rapid engineering of complex antibiotic assembly lines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Li Thong ◽  
Yingxin Zhang ◽  
Ying Zhuo ◽  
Katherine J. Robins ◽  
Joanna K. Fyans ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Chemical Synthesis ◽  
Gene Editing ◽  
Gene Knockout ◽  
Substrate Selectivity ◽  
Assembly Lines ◽  
Bioactive Natural Products ◽  
Peptide Synthetases ◽  
New Antibiotics ◽  
Lipopeptide Antibiotic

AbstractRe-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

scholarly journals Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering

2021 ◽  
Author(s):  
Nghi Dang ◽  
Alissa Lance-Byrne ◽  
Angela Tung ◽  
Xiaoge Guo ◽  
Ryan J Cecchi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Genome Engineering ◽  
Gene Knockout ◽  
Transient Transfection ◽  
Cell Engineering ◽  
Genetic Manipulations ◽  
Expression Plasmids

Abstract CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.

scholarly journals Unsuccessful attempt at gene-editing by homologous recombination in the zebrafish germ line using the approach of “Rong and Golic”

2012 ◽  
Vol 21 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Rosalind Brookfield ◽  
Felix Dafhnis-Calas ◽  
Zhengyao Xu ◽  
William Brown
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Germ Line ◽  
Unsuccessful Attempt

scholarly journals Advances in Enhanced Menaquinone-7 Production From Bacillus subtilis

2021 ◽  
Vol 9 ◽  
Author(s):  
Chaoyong Liao ◽  
Hammed Ayansola ◽  
Yanbo Ma ◽  
Koichi Ito ◽  
Yuming Guo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chemical Synthesis ◽  
Biosynthetic Pathway ◽  
Gene Editing ◽  
Vitamin K2 ◽  
Future Studies ◽  
Biofilm Reactors ◽  
Nutraceutical Compounds ◽  
Activation System ◽  
Crispr Activation

The production of nutraceutical compounds through biosynthetic approaches has received considerable attention in recent years. For example, Menaquinone-7 (MK-7), a sub-type of Vitamin K2, biosynthesized from Bacillus subtilis (B. subtilis), proved to be more efficiently produced than the conventional chemical synthesis techniques. This is possible due to the development of B. subtilis as a chassis cell during the biosynthesis stages. Hence, it is imperative to provide insights on the B. subtilis membrane permeability modifications, biofilm reactors, and fermentation optimization as advanced techniques relevant to MK-7 production. Although the traditional gene-editing method of homologous recombination improves the biosynthetic pathway, CRISPR-Cas9 could potentially resolve the drawbacks of traditional genome editing techniques. For these reasons, future studies should explore the applications of CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) system gene-editing tools in the MK-7 anabolism pathway.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing efficiency

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yuki Yoshino ◽  
Shino Endo ◽  
Zhenghao Chen ◽  
Huicheng Qi ◽  
Gou Watanabe ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Recombination Activity ◽  
Site Specific ◽  
Editing Efficiency

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination

2020 ◽  
Vol 98 (3) ◽  
pp. 314-326 ◽  
Author(s):  
Kathleen M. Attwood ◽  
Jayme Salsman ◽  
Dudley Chung ◽  
Sabateeshan Mathavarajah ◽  
Carter Van Iderstine ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Genotoxic Stress ◽  
Nuclear Bodies ◽  
Homology Directed Repair ◽  
Pml Nuclear Bodies ◽  
Promyelocytic Leukemia Nuclear Bodies ◽  
Chromosomal Break ◽  
The Impact

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

scholarly journals Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

2019 ◽  
Vol 48 (2) ◽  
pp. 517-532 ◽  
Author(s):  
Bin Liu ◽  
Siwei Chen ◽  
Anouk La Rose ◽  
Deng Chen ◽  
Fangyuan Cao ◽  
...  
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Rapid Development ◽  
Open Chromatin ◽  
Chromatin Compaction ◽  
Histone Deacetylase 1 ◽  
Cas9 Protein ◽  
Target Dna ◽  
Histone Modifiers ◽  
Low Efficiency

Abstract Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

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