Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

Ling, Xinyu; Xie, Bingteng; Gao, Xiaoqin; Chang, Lei; Wei, Zheng; Chen, Heqi; Huang, Yujia; Tan, Liansheng; Mo, Li; Liu, Tao

doi:10.1126/sciadv.aaz0051

Cited by 85 publications

(63 citation statements)

References 41 publications

(43 reference statements)

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“…With regard to the former, our detection method of biolistic delivery in pollen and pollinated pistils would contribute to increasing the efficiency at various reproductive steps. In the latter regard, a number of approaches aimed at enhancing the efficiency of genome editing have been reported, including the modification of Cas9 (Ling et al 2020; Osakabe et al 2020), design of guide RNA (Moon et al 2019), the use of RNPs (Liang et al 2018; Svitashev et al 2016), and the use of small chemical compounds (Yu et al 2015). Various selection methods have also been reported, including those based on antibiotic resistance (Chesnokov and Manteuffel 2000) and herbicide resistance, by targeting endogenous genes (Han and Kim 2019).…”

Section: Discussionmentioning

confidence: 99%

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Nagahara

Higashiyama

Mizuta

2021

Preprint

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In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

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Section: Discussionmentioning

confidence: 99%

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Nagahara

Higashiyama

Mizuta

2021

Preprint

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“…This strategy was shown to facilitate HDR events while mitigating undesired NHEJ edits in human immortalized and stem cells (84,85). A more recent strategy combined a chemically modified Cas9 to the ssODN donor or a DNA adaptor that recruits the donor template, either of which improved HDR efficiency by localizing the donor template near the cleavage site (86). Despite these advancements, HDR is still achieved at a relatively low efficiency in eukaryotic cells and use of relatively harmful agents in cells such as NHEJ chemical inhibitors may not be ideal in a clinical setting.…”

Section: Precision Gene Editing With Crisprmentioning

confidence: 99%

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

2020

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A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.

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“…Furthermore, non-chemically modified ssODNs can be used. The authors report a 10-fold increase in HDR in HEK293 cells and a three-fold increase in mouse zygotes [ 117 ]. While these strategies have yet to be tested in HSPCs for diseases such as sickle cell, where large gene insertions are not required, this may prove to be beneficial.…”

Section: Strategies To Improve Hdr-mediated Gene Editing In Hspcsmentioning

confidence: 99%

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

Salisbury-Ruf

Larochelle

2021

JCM

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Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) is a promising strategy for the treatment of inherited blood disorders, obviating many of the limitations associated with viral vector-mediated gene therapies. The use of CRISPR/Cas9 or other programmable nucleases and improved methods of homology template delivery have enabled precise ex vivo gene editing. These transformative advances have also highlighted technical challenges to achieve high-efficiency gene editing in HSPCs for therapeutic applications. In this review, we discuss recent pre-clinical investigations utilizing homology-mediated gene editing in HSPCs and highlight various strategies to improve editing efficiency in these cells.

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Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

Cited by 85 publications

References 41 publications

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

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