scholarly journals Efficient Homology-directed Repair with Circular ssDNA Donors

2019 ◽  
Author(s):  
Sukanya Iyer ◽  
Aamir Mir ◽  
Joel Vega-Badillo ◽  
Benjamin P. Roscoe ◽  
Raed Ibraheim ◽  
...  
Keyword(s):  
Genome Editing ◽  
K562 Cells ◽  
Cost Effective ◽  
Cell Types ◽  
Traffic Light ◽  
Homology Directed Repair ◽  
Mammalian Cell Lines ◽  
Cost Effective Method ◽  
Circular Ssdna ◽  
Fluorescent Tag

AbstractWhile genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

scholarly journals MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

2019 ◽  
Author(s):  
Cassidy Petree ◽  
Gaurav K Varshney
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Fold Increase ◽  
Targeted Mutagenesis ◽  
Model Organisms ◽  
Multiple Alleles ◽  
Cost Effective Method ◽  
Single Reaction

AbstractThe powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.

scholarly journals Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair

2020 ◽  
Vol 48 (7) ◽  
pp. e38-e38 ◽  
Author(s):  
Sara E DiNapoli ◽  
Raul Martinez-McFaline ◽  
Caitlin K Gribbin ◽  
Paul J Wrighton ◽  
Courtney A Balgobin ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Cell Types ◽  
Specific Cell ◽  
Loss Of Function ◽  
Genomic Deletions ◽  
Homology Directed Repair ◽  
Linear Dsdna ◽  
Rapid Generation

Abstract CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

scholarly journals Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

2020 ◽  
Author(s):  
Benedetta Artegiani ◽  
Delilah Hendriks ◽  
Joep Beumer ◽  
Rutger Kok ◽  
Xuan Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Intestinal Cell ◽  
Precise Integration ◽  
Exogenous Dna ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Efficient Generation

AbstractCRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homology-independent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

scholarly journals 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

2018 ◽  
Author(s):  
Krishna S. Ghanta ◽  
Gregoriy A. Dokshin ◽  
Aamir Mir ◽  
Pranathi Meda Krishnamurthy ◽  
Hassan Gneid ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Genetic Basis ◽  
Cell Types ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach that directly addresses the underlying genetic basis of disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit the number of donor molecules available to the HDR machinery, thus limiting HDR efficacy. Here, we explore modifications to both double-stranded and single-stranded repair template DNAs and describe simple 5′ end modifications that consistently and dramatically increase donor potency and HDR efficacy across cell types and species.

scholarly journals Efficient Immune Cell Genome Engineering with Improved CRISPR Editing Tools

2020 ◽  
Author(s):  
Waipan Chan ◽  
Rachel A. Gottschalk ◽  
Yikun Yao ◽  
Joel L. Pomerantz ◽  
Ronald N. Germain
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Genome Engineering ◽  
Immune Cell ◽  
Animal Experiments ◽  
Adaptive Immune System ◽  
Cell Types ◽  
Acid Activation ◽  
Homology Directed Repair ◽  
Cell Genome

AbstractCRISPR (clustered regularly interspaced short palindromic repeats)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of sensitive intracellular sensors. Likewise, lymphocytes of the adaptive immune system are largely refractory to CRISPR-enhanced homology-directed repair (HDR) due to inefficient or toxic delivery of donor templates via transient transfection methods. To overcome these challenges and limitations, we developed three improved methods for CRISPR-based genome editing using a hit-and-run transient expression strategy to minimize off-target effects and generate more precise genome editing. Overall, our enhanced CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering are expected to be widely applicable not only in hematopoietic cells but also other mammalian cell types of interest.All animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee.

scholarly journals A total synthetic approach to CRISPR/Cas9 genome editing and homology directed repair

2018 ◽  
Author(s):  
Sara E. DiNapoli ◽  
Raul Martinez-McFaline ◽  
Caitlin K. Gribbin ◽  
Paul Wrighton ◽  
Courtney A. Balgobin ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Cell Types ◽  
Specific Cell ◽  
Synthetic Approach ◽  
Loss Of Function ◽  
Dna Templates ◽  
Homology Directed Repair

ABSTRACTCRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

scholarly journals Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

2017 ◽  
Author(s):  
Eric J. Aird ◽  
Klaus N. Lovendahl ◽  
Amber St. Martin ◽  
Reuben S. Harris ◽  
Wendy R. Gordon
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Repair Efficiency ◽  
Rnp Complex ◽  
Multiple Cell ◽  
Low Efficiency ◽  
A Site ◽  
Protein Tags

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome where the Cas9 nuclease then induces a double stranded break (DSB)1,2. The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB3,4. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded donor oligonucleotide (ssODN) to the Cas9/guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease5, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to an 8-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci, and is more pronounced at low RNP concentrations.

CAD Of Anticorrosive Protection Of Steel Structures

2019 ◽  
pp. 18-23
Keyword(s):  
Protective Coating ◽  
Computer Aided Design ◽  
Steel Structures ◽  
Cost Effective ◽  
Operating Conditions ◽  
Computational Tool ◽  
Protection Method ◽  
Cost Effective Method ◽  
The Cost ◽  
Aided Design

The choice of cost-effective method of anticorrosive protection of steel structures is an urgent and time consuming task, considering the significant number of protection ways, differing from each other in the complex of technological, physical, chemical and economic characteristics. To reduce the complexity of solving this problem, the author proposes a computational tool that can be considered as a subsystem of computer-aided design and used at the stage of variant and detailed design of steel structures. As a criterion of the effectiveness of the anti-corrosion protection method, the cost of the protective coating during the service life is accepted. The analysis of existing methods of steel protection against corrosion is performed, the possibility of their use for the protection of the most common steel structures is established, as well as the estimated period of effective operation of the coating. The developed computational tool makes it possible to choose the best method of protection of steel structures against corrosion, taking into account the operating conditions of the protected structure and the possibility of using a protective coating.

Monitoring effluent quality in hypertrophic wastewater reservoirs using remote sensing

1996 ◽  
Vol 33 (8) ◽  
pp. 23-29 ◽  
Author(s):  
I. Dor ◽  
N. Ben-Yosef
Keyword(s):  
Water Quality ◽  
Theoretical Model ◽  
Principal Component ◽  
Cost Effective ◽  
Operational Parameters ◽  
Effluent Quality ◽  
Optical Images ◽  
Routine Monitoring ◽  
Cost Effective Method ◽  
Wastewater Quality

About one hundred and fifty wastewater reservoirs store effluents for irrigation in Israel. Effluent qualities differ according to the inflowing wastewater quality, the degree of pretreatment and the operational parameters. Certain aspects of water quality like concentration of organic matter, suspended solids and chlorophyll are significantly correlated with the water column transparency and colour. Accordingly optical images of the reservoirs obtained from the SPOT satellite demonstrate pronounced differences correlated with the water quality. The analysis of satellite multispectral images is based on a theoretical model. The model calculates, using the radiation transfer equation, the volume reflectance of the water body. Satellite images of 99 reservoirs were analyzed in the chromacity space in order to classify them according to water quality. Principal Component Analysis backed by the theoretical model increases the method sensitivity. Further elaboration of this approach will lead to the establishment of a time and cost effective method for the routine monitoring of these hypertrophic wastewater reservoirs.

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