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Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts
Genome Research ( IF 6.2 ) Pub Date : 2024-12-03 , DOI: 10.1101/gr.279479.124
Irmgard U. Haussmann, Thomas C. Dix, David W.J. McQuarrie, Veronica Dezi, Abdullah I. Hans, Roland Arnold, Matthias Soller

A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N6 methylated adenosines (m6A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR–Cas9 genome editing advance the efficient use of this technique for safer applications in humans.

中文翻译:


使用 PlatinumCRISPr 进行结构优化的 sgRNA 筛选,用于高效的 Cas9 基因敲除



单向导 RNA (sgRNA) 指导 Cas9 核酸酶对双链 DNA 进行基因特异性切割。高 Cas9 活性对于有效的基因编辑至关重要,以通过同源重组产生基因缺失和基因替换。然而,在人类细胞培养筛选或模式生物中,超过一半的随机选择的 sgRNA 序列的切割效率低于 50%。我们使用体外测定来确定最大 sgRNA 活性的内在分子参数,包括 sgRNA 的正确折叠和 Cas9 结构信息。从对 10 多个数据集的比较中,我们发现 sgRNA 设计中的主要限制源于 sgRNA 的二级结构缺陷、种子区域的序列上下文、GC 上下文和有害基序,但我们也发现当应用于不同的数据集时,不同的预测工具之间存在相当大的差异。为了帮助选择高效的 sgRNA,我们开发了基于 Web 的 PlatinumCRISPr,这是一种 sgRNA 设计工具,用于评估碱基配对和序列组成参数,以优化设计用于 Cas9 基因组编辑的高效 sgRNA。我们应用该工具来选择 sgRNA,以在果蝇 Ythdc1Ythdf 中有效地产生基因缺失,这些基因缺失与 mRNA 中的 N6 甲基化腺苷 (m6A) 结合。然而,我们发现用 sgRNA 和 Cas9 产生小缺失会导致缺失的 DNA 片段异位重新插入基因组中的其他位置。这些插入可以通过标准基因重组和染色体交换去除。这些对 sgRNA 设计和 CRISPR-Cas9 基因组编辑机制的新见解促进了该技术在人类中更安全的应用。
更新日期:2024-12-04
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