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Abstract：
CONCLUSIONS: Multiple variables that control the relative levels of successful heritable plant genome editing were addressed using simple case studies in Arabidopsis thaliana. The recent advent of genome editing technologies (especially CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats) has revolutionized various fields of scientific research. The process is much more specific than previous mutagenic processes and allows for targeting of nearly any gene of interest for the creation of loss-of-function mutations and many other types of editing, including gene-replacement and gene activation. However, not all CRISPR construct designs are successful, due to several factors, including differences in the strength and cell- or tissue-type specificity of the regulatory elements used to express the Cas9 (CRISPR Associated protein 9) DNA nuclease and single guide RNA components, and differences in the relative editing efficiency at different target areas within a given gene. Here we compare the levels of editing created in Arabidopsis thaliana by CRISPR constructs containing either different promoters, or altered target sites with varied levels of guanine-cytosine base content. Additionally, nuclease activity at sites targeted by imperfectly matched single guide RNAs was observed, suggesting that while the primary goal of most CRISPR construct designs is to achieve rapid, robust, heritable gene editing, the formation of unintended mutations at other genomic loci must be carefully monitored.
摘要：
结论：使用拟南芥中的简单案例研究解决了控制成功的可遗传植物基因组编辑相对水平的多个变量。最近出现的基因组编辑技术（特别是CRISPR，聚集的定期间隔短回文重复）彻底改变了科学研究的各个领域。该过程比以前的诱变过程更具特异性，并且允许靶向几乎任何感兴趣的基因，以创建功能丧失突变和许多其他类型的编辑，包括基因置换和基因激活。然而,并非所有的CRISPR结构设计都是成功的，由于几个因素，包括用于表达Cas9（CRISPR相关蛋白9）DNA核酸酶和单向导RNA成分的调节元件的强度和细胞或组织类型特异性的差异，以及给定基因内不同目标区域的相对编辑效率的差异。在这里，我们比较了包含不同启动子的CRISPR构建体在拟南芥中创建的编辑水平，或改变了具有不同水平的鸟嘌呤-胞嘧啶碱基含量的靶位点。此外,观察到不完全匹配的单向导RNA靶向位点的核酸酶活性，这表明，虽然大多数CRISPR构建体设计的主要目标是实现快速，健壮,遗传基因编辑，必须仔细监测其他基因组位点意外突变的形成.
