用于基因编辑应用的紧凑型 CRISPR-Cas12f 病毒递送。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2024-06-01 Epub Date: 2024-05-02 DOI:10.1089/crispr.2024.0010
Allison Sharrar, Zuriah Meacham, Johanna Staples-Ager, Luisa Arake de Tacca, David Rabuka, Trevor Collingwood, Michael Schelle
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引用次数: 0

摘要

要在体内治疗人类遗传病,就必须将 CRISPR 基因编辑机器有效地输送到受影响的细胞和器官。基因编辑领域在体外疗法和利用脂质纳米粒子技术向肝脏体内递送方面取得了临床进展。腺相关病毒(AAV)血清型已被发现并设计用于向人体几乎所有器官输送遗传物质。然而,大多数 CRISPR-Cas 系统体积庞大,限制了病毒基因组的包装,降低了药物开发的灵活性和生产效率。在这里,我们展示了使用微型CRISPR-Cas12f系统进行高效CRISPR基因编辑的方法,该系统将扩大的基因组靶向打包到AAV颗粒中。我们为四个治疗基因靶点确定了高效的向导,并将向导和 Cas12f 核酸酶编码到单个 AAV 中。然后,我们在多个细胞系、患者成纤维细胞和原代肝细胞中演示了编辑。然后,我们对细胞进行了脱靶编辑筛选,证明了疗法的安全性。这些成果是将 CRISPR 编辑应用于体内不同基因序列和器官的重要一步。
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Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications.

Treating human genetic conditions in vivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with in vivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduces drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying CRISPR editing to diverse genetic sequences and organs in the body.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
期刊最新文献
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