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Updated: May 23, 2025

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

通过解开细胞CRISPR裂变结果,对合成gRNA活动和神秘DNA修复进行可靠的预测

Stephan Riesenberg¹, Philipp Kanis², Rosa Karlic³

¹Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. stephan_riesenberg@eva.mpg.de.

Nature communications

|May 21, 2025

概括

预测导向RNA (gRNA) 活动对于CRISPR基因编辑至关重要. 这项研究引入了一种线性模型,以准确预测跨细胞系的合成gRNA效率,改进CRISPR应用.

科学领域:

分子生物学分子生物学
遗传学是一个遗传学.
生物技术是生物技术.

背景情况:

预测导向RNA (gRNA) 活动对于高效的CRISPR应用至关重要.
目前的方法通常依赖于预先选gRNAs,这是耗时的.
量化DNA插入和删除 (indels) 是一种常见但可能被低估的gRNA活性指标.

研究的目的:

评估化学合成的Cas9 gRNAs对各种DNA分裂结果的影响.
开发一个可靠的模型来预测合成gRNA活动.
评估Cas9和Cas12a基因编辑中大规模修复事件的频率.

主要方法:

用化学合成的Cas9 gRNAs来评估DNA裂变的结果.
开发了一个简单的线性模型来预测合成gRNA活动.
对 Cas9 和 Cas12a 系统分析了同质导向修复 (HDR) 的效率.

主要成果:

发现不同合成gRNAs的活性在很大程度上是相似的,并且在仅测量indels时经常被低估.
开发的线性模型可靠地预测不同细胞系和数据集的合成gRNA活动.
意外的大规模修复事件在Cas9中很常见,但不是Cas12a.

结论:

合成gRNA活动的预测模型通过减少经验查的需求来增强CRISPR应用.
Cas12a可能为基因疗法提供比Cas9更安全的替代品,因为意外的大规模修复事件较少.

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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

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Published on: November 1, 2024

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