Methodologies for Improving HDR Efficiency

Liu, Mingjie; Rehman, Saad; Tang, Xidian; Gu, Ke; Fan, Qin-Lei; Chen, Dekun; Ma, Wentao

doi:10.3389/fgene.2018.00691

Cited by 248 publications

(200 citation statements)

References 122 publications

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“…The ability to control DSB repair pathway choice has been long sought after, with numerous different tools and techniques developed to influence repair choice 4,5,9 . The data shown here demonstrates that the first ever example of a tool successfully bypassing the rate limiting step of HR repair.…”

Section: Discussionmentioning

confidence: 99%

A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

Hackley

2020

Preprint

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A novel Cas9 fusion protein designed to increase HDR rates through bypassing the rate limiting step of homologous recombination repair. Abstract:CRISPR/Cas9 has revolutionized genetic engineering, however, the inability to control double strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice, however particularly in the case of larger edits, none have been able to bypass the rate-limiting step of Homologous Recombination (HR): long-range 5' end resection. Here we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Cas9-HRs can increase the rate HR by 2-2.5 fold and decrease cellular toxicity by 2-4 fold compared to Cas9 in mammalian cells with minimal apparent editing site bias, thus making Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.

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Section: Discussionmentioning

confidence: 99%

A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

Hackley

2020

Preprint

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“…Alternatively, combo systems, such as the "nanoblade" platform, may represent suitable alternatives to deliver the DNA donor template, the Cas9 protein and the sgRNA [91]. Finally, while the recombination efficiency is often low, chemical and genetic modulation might help to promote the HDR pathway [92].…”

Section: Current Challengesmentioning

confidence: 99%

Towards Physiologically and Tightly Regulated Vectored Antibody Therapies

Page

Fusil

Cosset

2020

Cancers

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Cancers represent highly significant health issues and the options for their treatment are often not efficient to cure the disease. Immunotherapy strategies have been developed to modulate the patient’s immune system in order to eradicate cancerous cells. For instance, passive immunization consists in the administration at high doses of exogenously produced monoclonal antibodies directed either against tumor antigen or against immune checkpoint inhibitors. Its main advantage is that it provides immediate immunity, though during a relatively short period, which consequently requires frequent injections. To circumvent this limitation, several approaches, reviewed here, have emerged to induce in vivo antibody secretion at physiological doses. Gene delivery vectors, such as adenoviral vectors or adeno-associated vectors, have been designed to induce antibody secretion in vivo after in situ cell modification, and have driven significant improvements in several cancer models. However, anti-idiotypic antibodies and escape mutants have been detected, probably because of both the continuous expression of antibodies and their expression by unspecialized cell types. To overcome these hurdles, adoptive transfer of genetically modified B cells that secrete antibodies either constitutively or in a regulated manner have been developed by ex vivo transgene insertion with viral vectors. Recently, with the emergence of gene editing technologies, the endogenous B cell receptor loci of B cells have been modified with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas-9) system to change their specificity in order to target a given antigen. The expression of the modified BCR gene hence follows the endogenous regulation mechanisms, which may prevent or at least reduce side effects. Although these approaches seem promising for cancer treatments, major questions, such as the persistence and the re-activation potential of these engineered cells, remain to be addressed in clinically relevant animal models before translation to humans.

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“…Although the HDR (homology directed repair) knock-in approach appears to suffer from low efficiency when used in non-traditional insect models (C. M. Clark-Hachtel, K. D. Deem and Y. Tomoyasu, unpublished data), various strategies have been developed to improve success rates (e.g. Aird et al, 2018;Savic et al, 2018, also see Bier et al, 2018Liu et al, 2019 for review of additional methods to increase the HDR efficiency), some of which might be worth pursuing when performing HDR knock-in in insects. In addition, NHEJ knock-in (Auer et al, 2014;Watanabe et al, 2017), including the CRISPaint (CRISPR-assisted insertion tagging) system (Bosch et al, 2020;Schmid-Burgk et al, 2016), or MMEJ (microhomology mediated end joining) knock-in (e.g.…”

Section: Functional Analysis Of Enhancers Through Genome Editingmentioning

confidence: 99%

How to study enhancers in non-traditional insect models

Tomoyasu

Halfon

2020

Journal of Experimental Biology

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Transcriptional enhancers are central to the function and evolution of genes and gene regulation. At the organismal level, enhancers play a crucial role in coordinating tissue-and context-dependent gene expression. At the population level, changes in enhancers are thought to be a major driving force that facilitates evolution of diverse traits. An amazing array of diverse traits seen in insect morphology, physiology and behavior has been the subject of research for centuries. Although enhancer studies in insects outside of Drosophila have been limited, recent advances in functional genomic approaches have begun to make such studies possible in an increasing selection of insect species. Here, instead of comprehensively reviewing currently available technologies for enhancer studies in established model organisms such as Drosophila, we focus on a subset of computational and experimental approaches that are likely applicable to non-Drosophila insects, and discuss the pros and cons of each approach. We discuss the importance of validating enhancer function and evaluate several possible validation methods, such as reporter assays and genome editing. Key points and potential pitfalls when establishing a reporter assay system in non-traditional insect models are also discussed. We close with a discussion of how to advance enhancer studies in insects, both by improving computational approaches and by expanding the genetic toolbox in various insects. Through these discussions, this Review provides a conceptual framework for studying the function and evolution of enhancers in non-traditional insect models.

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Methodologies for Improving HDR Efficiency

Cited by 248 publications

References 122 publications

A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

Towards Physiologically and Tightly Regulated Vectored Antibody Therapies

How to study enhancers in non-traditional insect models

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