A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation.
A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation.
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CRIS/CAS9活性的生物物理模型，用于基因组编辑和基因调节的合理设计。
DOI：
10.1371/journal.pcbi.1004724
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发表时间：
2016-01
影响因子：
4.3
通讯作者：
Salis HM
中科院分区：
文献类型：
--
作者：
Farasat I;Salis HM
精确修改基因组和调节特定基因的能力将大大加速几个医学和工程应用。CRISPR/Cas9（II型）系统使用向导RNA结合和切割DNA，尽管控制其靶向和脱靶活性的变量仍然很难表征。在这里，我们开发并参数化了基于Cas9的基因组编辑和基因调控的全系统生物物理模型，以预测如何改变向导RNA序列，DNA超螺旋密度，Cas9和crRNA表达水平，生物体和生长条件以及实验条件共同控制所有DNA位点处基于dCas 9的结合和基于Cas9的切割的动力学与经典和非经典PAM。我们结合联合收割机统计热力学和动力学来建模Cas9：crRNA复合物形成、扩散、位点选择、可逆R环形成和切割，使用大量的结构、生化、表达和下一代测序数据来确定动力学参数并开发自由能模型。我们的研究结果将DNA超螺旋确定为控制Cas9结合的新机制。使用该模型，我们预测了整个噬菌体和人类基因组中Cas9的脱靶结合频率，并解释了为什么Cas9的脱靶活性如此之高。有了这种更好的理解，我们提出了几个规则，设计实验，以尽量减少脱靶活动。我们还讨论了工程dCas 9为基础的遗传电路的影响。CRISPR/Cas9免疫系统有可能彻底改变医学和生物技术，使研究人员能够在精确的位置切割生物体的基因组DNA。虽然Cas9可能是迄今为止开发的最通用和最易于使用的基因治疗技术，但它并不完美;该酶还可以在生物体基因组中不需要的位置切割DNA。Cas9的脱靶活性必须大大减少，以进一步提高其效用。在这里，我们开发了一个全系统的定量物理模型，以更好地了解共同控制Cas9脱靶切割的所有因素。我们使用来自我们实验室的基因调控数据以及来自其他实验室的结构，生物化学和下一代测序数据来解决未知参数。在几个例子中使用该模型，我们解释了Cas9如何根据向导RNA序列、Cas9和crRNA表达水平、生物体的基因组和生物体的细胞生长速率来识别靶向与脱靶DNA位点。然后，我们提出了几个规则，设计实验最小的脱靶活动。
The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. The CRISPR/Cas9 immunity system has the potential to revolutionize medicine and biotechnology by enabling researchers to cut an organism’s genomic DNA at precise locations. While Cas9 is perhaps the most versatile and easy-to-use technique for gene therapy developed yet, it is not perfect; the enzyme can also cut DNA at unwanted locations in an organism’s genome. Cas9’s off-target activity must be greatly minimized to further improve its utility. Here, we develop a system-wide, quantitative, physical model to better understand all the factors that collectively control Cas9’s off-target cleavage. We solve for the unknown parameters using gene regulation data from our laboratory as well as structural, biochemical, and next-generation sequencing data from other laboratories. Using the model in several examples, we explain how Cas9 identifies on-target versus off-target DNA sites, depending on the guide RNA sequence, the Cas9 and crRNA expression levels, the organism’s genome, and the organism’s cellular growth rate. We then propose several rules for designing experiments with minimal off-target activity.