Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

doi:10.1016/j.cjche.2021.02.009

中国化学工程学报 ›› 2021, Vol. 38 ›› Issue (10): 216-220.DOI: 10.1016/j.cjche.2021.02.009

• Biotechnology and Bioengineering • 上一篇下一篇

Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Xuanyu Li, Qiang Feng, Ziwei Han, Xingyu Jiang

Shenzhen Bay Laboratory, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen 518055, China

收稿日期:2020-04-17 修回日期:2021-01-29 出版日期:2021-10-28 发布日期:2021-12-02
通讯作者: Xingyu Jiang
基金资助:
Xuanyu Li, Qiang Feng and Ziwei Han contributed equally to this work. We thank the National Natural Science Foundation of China (21761142006, 21535001, and 81730051), Shenzhen Science and Technology Program (KQTD20190929172743294), the National Key R&D Program of China (2018YFA0902600), the Chinese Academy of Sciences (QYZDJ-SSW-SLH039), Shenzhen Bay Laboratory (SZBL2019062801004), Tencent Foundation through the XPLORER PRIZE for financial support.

Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Xuanyu Li, Qiang Feng, Ziwei Han, Xingyu Jiang

Shenzhen Bay Laboratory, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen 518055, China

Received:2020-04-17 Revised:2021-01-29 Online:2021-10-28 Published:2021-12-02
Contact: Xingyu Jiang
Supported by:
Xuanyu Li, Qiang Feng and Ziwei Han contributed equally to this work. We thank the National Natural Science Foundation of China (21761142006, 21535001, and 81730051), Shenzhen Science and Technology Program (KQTD20190929172743294), the National Key R&D Program of China (2018YFA0902600), the Chinese Academy of Sciences (QYZDJ-SSW-SLH039), Shenzhen Bay Laboratory (SZBL2019062801004), Tencent Foundation through the XPLORER PRIZE for financial support.

摘要/Abstract

摘要： Most non-viral carriers for in vitro delivery of nucleic acids suffer from low efficiency of introducing mRNA and other nucleic acids, especially large mRNA. Cas9 protein is the nuclease part of the powerful gene-editing tool, CRISPR/Cas9 system, Cas9 mRNA is particularly large, thus presents a big challenge for delivery. We assembled a multilayered biodegradable nanocarrier to load Cas9 mRNA inside to protect Cas9 mRNA from degradation. We used a microfluidic chip to synthesize a small, positively charged, and degradable core to attract negatively charged Cas9 mRNA. The microfluidic assembly allows the core to be small enough to incorporate into a cationic liposome. The multilayered nanocarriers elevated the delivery efficiency of Cas9 mRNA by over 2 folds and increased the expression by over 5 folds compared to commercially used non-viral carriers. In addition, the multilayered nanocarriers do not require reduced serum medium for transfection. When using the standard complete medium for transfection, the multilayered nanocarriers could increase the expression of Cas9 mRNA by over 15 folds compared to commercially used non-viral carriers. The co-delivery of Cas9 mRNA and sgRNA via LRC elevated the gene-editing efficiency by 3 folds compared to that via commercially used non-viral carriers. Based on the higher transfection efficiency of Cas9 mRNA/sgRNA than commercially used non-viral carriers, these multilayered nanocarriers may have a good prospect as efficient commercial delivery carriers for Cas9 mRNA/sgRNA and other nucleic acids.

关键词: Nanotechnology, Biomedical engineering, Microfluidics, CRISPR/Cas9 system, Poly(lactic-co-glycolic acid)

Abstract: Most non-viral carriers for in vitro delivery of nucleic acids suffer from low efficiency of introducing mRNA and other nucleic acids, especially large mRNA. Cas9 protein is the nuclease part of the powerful gene-editing tool, CRISPR/Cas9 system, Cas9 mRNA is particularly large, thus presents a big challenge for delivery. We assembled a multilayered biodegradable nanocarrier to load Cas9 mRNA inside to protect Cas9 mRNA from degradation. We used a microfluidic chip to synthesize a small, positively charged, and degradable core to attract negatively charged Cas9 mRNA. The microfluidic assembly allows the core to be small enough to incorporate into a cationic liposome. The multilayered nanocarriers elevated the delivery efficiency of Cas9 mRNA by over 2 folds and increased the expression by over 5 folds compared to commercially used non-viral carriers. In addition, the multilayered nanocarriers do not require reduced serum medium for transfection. When using the standard complete medium for transfection, the multilayered nanocarriers could increase the expression of Cas9 mRNA by over 15 folds compared to commercially used non-viral carriers. The co-delivery of Cas9 mRNA and sgRNA via LRC elevated the gene-editing efficiency by 3 folds compared to that via commercially used non-viral carriers. Based on the higher transfection efficiency of Cas9 mRNA/sgRNA than commercially used non-viral carriers, these multilayered nanocarriers may have a good prospect as efficient commercial delivery carriers for Cas9 mRNA/sgRNA and other nucleic acids.

Key words: Nanotechnology, Biomedical engineering, Microfluidics, CRISPR/Cas9 system, Poly(lactic-co-glycolic acid)

Xuanyu Li, Qiang Feng, Ziwei Han, Xingyu Jiang. Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics[J]. 中国化学工程学报, 2021, 38(10): 216-220.

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Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

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