A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.
A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.
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DOI：
10.1038/s41467-022-28994-2
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发表时间：
2022-03-15
影响因子：
16.6
通讯作者：
Depken M
中科院分区：
文献类型：
--
作者：
Eslami-Mossallam B;Klein M;Smagt CVD;Sanden KVD;Jones SK Jr;Hawkins JA;Finkelstein IJ;Depken M
S. pyogenes (Sp) Cas9内切酶是一种重要的基因编辑工具。SpCas9基于与复杂单导RNA （sgRNA）的互补性被定向到靶点。然而，SpCas9-sgRNA也仅以部分互补的方式结合和切割基因组脱靶。迄今为止，我们缺乏定量预测裂解和结合活性的能力，并且依赖于二元分类方案来识别强脱靶。我们报告了一个定量动力学模型，以自由能的形式捕获了spcas9介导的链取代反应。该模型预测结合和裂解活性作为时间，目标和实验条件的函数。经过高通量批量生化数据的训练和验证，我们的模型预测了最近在单分子实验中观察到的中间r环状态，以及相关的转化率。最后，我们表明，我们的定量活动预测器可以减少到一个二进制脱靶分类器，优于现有的最先进的技术。我们的方法是可扩展的，并且可以表征任何CRISPR-Cas核酸酶-对SpCas9的自然和未来高保真变体进行基准测试；阐明CRISPR保真度的决定因素；并揭示了在工程系统中提高特异性和效率的途径。Cas9脱靶位点可以通过许多生物信息学工具来预测。在这里，作者提出了以自由能形式表征SpCas9动力学的低复杂性机制模型，允许在散装生物化学，单分子和全基因组分析实验中定量预测脱靶活性。
The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease – benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems. Cas9 off-target sites can be predicted by many bioinformatics tools. Here the authors present low complexity mechanistic model that characterizes SpCas9 kinetics in free-energy terms, allowing quantitative prediction of off-target activity in bulk-biochemistry, single molecule, and whole-genome profiling experiments.
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影响因子：
12.3
作者：
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DOI：
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期刊：
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影响因子：
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作者：
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影响因子：
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