Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

Yoon, Ji-Young; Kwon, Daekee; Han, Mi-Jung; Choi, Sunmee; Park, Slki; Lee, Junghyuk; Lee, Kiwook; Lee, John J.; Lee, Seung‐Hee; Kang, Kyung‐Sun; Choe, Senyon

doi:10.1038/s41598-021-95406-8

Cited by 16 publications

(10 citation statements)

References 34 publications

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“…The efficacy of CRISPR-Cas9 systems for genome editing is typically correlated with their level of off-target modifications [82]: increased efficacy tends to come at the price of higher off-target mutation rates [82], i.e., there is a trade-off between efficacy and targeting precision, and the number of off-target modifications depends on the design and execution of the particular genome-editing approach. Not all genome-editing approaches are optimized for maximum targeting precision and thus a very low-level of off-target modifications.…”

Section: Considerations Concerning the Assessment Of Unintended Genet...mentioning

confidence: 99%

Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU

Eckerstorfer

Dolezel

Engelhard

et al. 2023

Plants

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The current initiative of the European Commission (EC) concerning plants produced using certain new genomic techniques, in particular, targeted mutagenesis and cisgenesis, underlines that a high level of protection for human and animal health and the environment needs to be maintained when using such applications. The current EU biosafety regulation framework ensures a high level of protection with a mandatory environmental risk assessment (ERA) of genetically modified (GM) products prior to the authorization of individual GMOs for environmental release or marketing. However, the guidance available from the European Food Safety Authority (EFSA) for conducting such an ERA is not specific enough regarding the techniques under discussion and needs to be further developed to support the policy goals towards ERA, i.e., a case-by-case assessment approach proportionate to the respective risks, currently put forward by the EC. This review identifies important elements for the case-by-case approach for the ERA that need to be taken into account in the framework for a risk-oriented regulatory approach. We also discuss that the comparison of genome-edited plants with plants developed using conventional breeding methods should be conducted at the level of a scientific case-by-case assessment of individual applications rather than at a general, technology-based level. Our considerations aim to support the development of further specific guidance for the ERA of genome-edited plants.

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Section: Considerations Concerning the Assessment Of Unintended Genet...mentioning

confidence: 99%

Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU

Eckerstorfer

Dolezel

Engelhard

et al. 2023

Plants

View full text Add to dashboard Cite

show abstract

“…Fusion with chromatinmodulating peptides has been demonstrated to improve the editing efficiency of SpCas9 by up to several fold [28]. Other attempts to enhance the efficiency of Cas-mediated gene editing include fusion of SpCas9 to proteins, including exonucleases, endonucleases, and deaminases [29]. For example, chimeric fusion with a three prime repair exonuclease 2 increased the mutagenic efficiency of SpCas9 2.5-fold [30].…”

Section: Editing Efficiencymentioning

confidence: 99%

Challenges to Gene Editing Approaches in the Retina

Bonillo

Pfromm

Fischer

2022

Klin Monbl Augenheilkd

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Retinal gene therapy has recently been at the cutting edge of clinical development in the diverse field of genetic therapies. The retina is an attractive target for genetic therapies such as gene editing due to the distinctive anatomical and immunological features of the eye, known as immune privilege, so that inherited retinal diseases (IRDs) have been studied in several clinical studies. Thus, rapid strides are being made toward developing targeted treatments for IRDs. Gene editing in the retina faces a group of heterogenous challenges, including editing efficiencies, off-target effects, the anatomy of the target organ, immune responses, inactivation, and identifying optimal application methods. As clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) based technologies are at the forefront of current gene editing advances, their specific editing efficiency challenges and potential off-target effects were assessed. The immune privilege of the eye reduces the likelihood of systemic immune responses following retinal gene therapy, but possible immune responses must not be discounted. Immune responses to gene editing in the retina may be humoral or cell mediated, with immunologically active cells, including microglia, implicated in facilitating possible immune responses to gene editing. Immunogenicity of gene therapeutics may also lead to the inactivation of edited cells, reducing potential therapeutic benefits. This review outlines the broad spectrum of potential challenges currently facing retinal gene editing, with the goal of facilitating further advances in the safety and efficacy of gene editing therapies.

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“…Although these reports demonstrated the positive effects of UCP1-expressing adipocytes on metabolism, technologies used for gene regulation have limitations. CRISPR gene editing has low efficiency and off-target effects, and lentiviral vectors integrate into the genome and alter gene transcription [ 19 , 20 , 21 , 22 ]. To improve the development of UCP1-expressing adipocytes for clinical translation, we suggest CRISPRa technology, delivered by baculovirus vectors.…”

Section: Introductionmentioning

confidence: 99%

Transplantation of Adipose-Tissue-Engineered Constructs with CRISPR-Mediated UCP1 Activation

Michurina

Stafeev

Boldyreva

et al. 2023

IJMS

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Thermogenic adipocytes have potential utility for the development of approaches to treat type 2 diabetes and obesity-associated diseases. Although several reports have proved the positive effect of beige and brown adipocyte transplantation in obese mice, translation to human cell therapy needs improvement. Here, we describe the application of CRISPR activation (CRISPRa) technology for generating safe and efficient adipose-tissue-engineered constructs with enhanced mitochondrial uncoupling protein 1 (UCP1) expression. We designed the CRISPRa system for the activation of UCP1 gene expression. CRISPRa-UCP1 was delivered into mature adipocytes by a baculovirus vector. Modified adipocytes were transplanted in C57BL/6 mice, followed by analysis of grafts, inflammation and systemic glucose metabolism. Staining of grafts on day 8 after transplantation shows them to contain UCP1-positive adipocytes. Following transplantation, adipocytes remain in grafts and exhibit expression of PGC1α transcription factor and hormone sensitive lipase (HSL). Transplantation of CRISPRa-UCP1-modified adipocytes does not influence glucose metabolism or inflammation in recipient mice. We show the utility and safety of baculovirus vectors for CRISPRa-based thermogenic gene activation. Our findings suggest a means of improving existing cell therapy approaches using baculovirus vectors and CRISPRa for modification and transplantation of non-immunogenic adipocytes.

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Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

Cited by 16 publications

References 34 publications

Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU

Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU

Challenges to Gene Editing Approaches in the Retina

Transplantation of Adipose-Tissue-Engineered Constructs with CRISPR-Mediated UCP1 Activation

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