Guide-specific loss of efficiency and off-target reduction with Cas9 variants.
Guide-specific loss of efficiency and off-target reduction with Cas9 variants.
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DOI：
10.1093/nar/gkad702
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发表时间：
2023-10-13
影响因子：
14.9
通讯作者：
Xu H
中科院分区：
文献类型：
--
作者：
Zhang L;He W;Fu R;Wang S;Chen Y;Xu H
高保真成簇规则间隔回文重复序列 (CRISPR) 相关蛋白 9 (Cas9) 变体已被开发出来，以降低 CRISPR 系统的脱靶效应，但代价是效率损失。为了系统地评估 Cas9 变体与不同单引导 RNA (sgRNA) 复合物的效率和脱靶耐受性，我们应用高通量活力筛选和合成配对 sgRNA-靶标系统来评估数千个 sgRNA 与两种高保真 Cas9 变体 HiFi 和 LZ3 的组合。将这些变体与野生型 SpCas9 进行比较，我们发现~20% 的 sgRNA 在与 HiFi 或 LZ3 复合时会导致效率显着损失。效率损失取决于 sgRNA 种子区域以及与 Cas9 REC3 结构域相互作用的非种子区域中第 15-18 位的序列背景，表明 REC3 结构域中的变体特异性突变是效率损失的原因。当不同的 sgRNA 与变体结合使用时，我们还观察到不同程度的序列依赖性脱靶减少。鉴于这些观察结果，我们开发了 GuideVar，这是一种基于迁移学习的计算框架，用于预测高保真变体的目标效率和脱靶效应。 GuideVar 有助于在 HiFi 和 LZ3 应用中确定 sgRNA 的优先级，正如使用这些高保真变体在高通量活力筛选中信噪比的改善所证明的那样。
High-fidelity clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA–target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against wild-type SpCas9, we found that ∼20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15–18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in the REC3 domain account for the loss of efficiency. We also observed various degrees of sequence-dependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer learning-based computational framework for the prediction of on-target efficiency and off-target effects with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.
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