Yuxiao Wang, Ruei-Zeng Lin, Meghan Harris, Bianca Lavayen, Neha Diwanji, Bruce McCreedy, Robert Hofmeister, Daniel Getts
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引用次数: 0
摘要
为了解决广泛的遗传疾病,基因组编辑工具是必要的,它可以在不引起双链断裂(dsb)或不需要DNA模板的情况下实现大基因的靶向递送。在这里,我们介绍了CRISPR- enabled Autonomous Transposable Element (CREATE),这是一种基因组编辑系统,将CRISPR/Cas9的可编程性和精确性与人类LINE-1 (L1)元件的rna介导的基因插入能力相结合。CREATE使用修饰的L1 mRNA携带有效载荷基因和Cas9缺口酶,以促进L1介导的逆转录和整合的靶向编辑,而不依赖于dsb或DNA模板。使用该系统,我们演示了将1.1 kb的基因表达盒可编程插入到人类细胞系和原代T细胞的特定基因组位点中。机制研究表明,CREATE编辑是高度特异性的,没有观察到脱靶事件。总之,这些发现表明CREATE是一种可编程的、基于rna的基因传递技术,具有广泛的治疗潜力。
CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.
To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-strand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE-1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using this system, we demonstrate programmable insertion of a 1.1 kb gene expression cassette into specific genomic loci of human cell lines and primary T cells. Mechanistic studies reveal that CREATE editing is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable, RNA-based gene delivery technology with broad therapeutic potential.
期刊介绍:
EMBO Reports is a scientific journal that specializes in publishing research articles in the fields of molecular biology, cell biology, and developmental biology. The journal is known for its commitment to publishing high-quality, impactful research that provides novel physiological and functional insights. These insights are expected to be supported by robust evidence, with independent lines of inquiry validating the findings.
The journal's scope includes both long and short-format papers, catering to different types of research contributions. It values studies that:
Communicate major findings: Articles that report significant discoveries or advancements in the understanding of biological processes at the molecular, cellular, and developmental levels.
Confirm important findings: Research that validates or supports existing knowledge in the field, reinforcing the reliability of previous studies.
Refute prominent claims: Studies that challenge or disprove widely accepted ideas or hypotheses in the biosciences, contributing to the correction and evolution of scientific understanding.
Present null data: Papers that report negative results or findings that do not support a particular hypothesis, which are crucial for the scientific process as they help to refine or redirect research efforts.
EMBO Reports is dedicated to maintaining high standards of scientific rigor and integrity, ensuring that the research it publishes contributes meaningfully to the advancement of knowledge in the life sciences. By covering a broad spectrum of topics and encouraging the publication of both positive and negative results, the journal plays a vital role in promoting a comprehensive and balanced view of scientific inquiry.