Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants: An Expanded Toolkit for Precision Genome Engineering
Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants: An Expanded Toolkit for Precision Genome Engineering
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植物中 CRISPR 相关 (Cas) 核酸酶的效率和特异性比较：精密基因组工程的扩展工具包
DOI：
10.1101/422766
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Raitskin O
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文献类型：
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作者：
Raitskin O
从细菌CRISPR（重复的调控间隔短回文重复序列）系统改编的用于适应性免疫的分子工具已广泛用于植物基因组工程，既用于研究基因功能又用于工程化期望的性状。现在使用许多不同的Cas（CRISPR相关）核酸酶，但是由于迄今为止进行的大多数研究使用各种植物物种和分子工具来工程化不同的靶标，因此难以得出关于不同核酸酶的比较性能的结论。由于再生工程植物所需的时间和精力，效率至关重要。此外，已经有几个报告的突变序列与目标不太完美的同一性。虽然在一些植物物种中，可以通过与亲本系回交来去除这些所谓的“脱靶”，但是当靶向密切相关的基因家族的特定成员时，基因组工程工具的特异性是重要的，特别是当最近的旁系同源物在基因组中共存并且不太可能分离时。特异性对于需要数年时间才能达到性成熟或克隆繁殖的物种也很重要。在这里，我们直接比较了来自不同细菌物种的Cas核酸酶的效率和特异性以及Cas9的工程变体。我们发现靶点的核苷酸含量与效率相关，并且来自金黄色葡萄球菌的Cas9（SaCas9）在诱导突变方面相对最有效。我们还证明了Cas9的“高保真”变体可以减少植物中的脱靶突变。我们将这些分子工具作为标准化的DNA部分，以促进其重复使用。
Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 fromStaphylococcus aureus(SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.