Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants: An Expanded Toolkit for Precision Genome Engineering

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory InterspacedShort Palindromic Repeats) systems for adaptive immunity have become widely usedfor plant genome engineering, both to investigate gene functions and to engineerdesirable traits. A number of different Cas (CRISPR-associated) nucleases are nowused but, as most studies performed to date have engineered different targets using avariety of plant species and molecular tools, it has been difficult to draw conclusionsabout the comparative performance of different nucleases. Due to the time and effortrequired to regenerate engineered plants, efficiency is critical. In addition, there havebeen several reports of mutations at sequences with less than perfect identity to thetarget. 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 isimportant when targeting specific members of closely-related gene families, especiallywhen 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 thatare clonally propagated. Here, we directly compare the efficiency and specificity of Casnucleases from different bacterial species together with engineered variants of Cas9.We find that the nucleotide content correlates with efficiency and that Cas9 fromStaphylococcus aureus is comparatively most efficient at inducing mutations. We alsodemonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations inplants. We present these molecular tools as standardised DNA parts to facilitate theirre-use.

源自细菌适应性免疫相关CRISPR（Clustered Regulatory Interspaced Short Palindromic Repeats）系统改造而来的分子工具，已被广泛应用于植物基因组工程，既可用于解析基因功能，也可用于培育优良性状。目前已涌现出多种不同的Cas（CRISPR-associated，CRISPR关联）核酸酶，但由于迄今为止的多数研究均依托不同植物物种与分子工具针对各异靶标开展编辑，因此难以直接比较不同核酸酶的编辑性能优劣。鉴于再生转基因植株需耗费大量时间与人力，编辑效率至关重要。此外，已有多项研究报道，在与靶标序列不完全匹配的位点也会产生突变。尽管在部分植物物种中，可通过回交亲本株系清除这类被称为‘脱靶位点（off-targets）’的突变，但当靶向紧密关联基因家族的特定成员时，基因组编辑工具的特异性仍尤为关键——尤其是当新近产生的旁系同源基因在基因组中位置相邻、且难以通过分离剔除时。对于需要多年才能达到性成熟，或是采用无性繁殖的物种而言，编辑特异性同样不容忽视。本研究直接比较了不同细菌来源的Cas核酸酶与多款Cas9改造变体的编辑效率与特异性。我们发现靶位点的核苷酸组成与编辑效率存在关联，且来自金黄色葡萄球菌（Staphylococcus aureus）的Cas9在诱导突变方面综合效率最高。同时我们证实，Cas9的‘高保真（high-fidelity）’变体可降低植物中的脱靶突变发生率。我们将这些分子工具标准化为DNA元件，以方便后续重复使用。