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利用 niper-cas9 在不损失目标活动的情况下通过定向进化, 最大限度地减少 crispr-cas9 的非目标效应
JoVE 신문
유전학
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JoVE 신문 유전학
Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
DOI:

11:37 min

February 26, 2019

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Chapters

  • 00:04Title
  • 01:45Positive and Negative Screening for Evolving Cas9
  • 04:47Delivery of Cas9 as an RNP with Truncated sgRNA
  • 06:17RNP Delivery
  • 07:27Transfection of Plasmids Encoding Sniper-Cas9 and sgRNA
  • 09:01Targeted Deep Sequencing for the Analysis of On-target and Potential Off-target Sites
  • 09:29Results: Analysis of Sniper-screen, On-target and Off-target Activities
  • 10:37Conclusion

Summary

자동 번역

在这里, 我们提出了一个优化 crispr-cas9 的协议, 以实现更高的特异性, 而不会丢失目标活动。我们使用一种称为狙击手屏幕的定向进化方法来查找具有所需特征的突变 cas9。sanicper-cas9 与截断的单导轨 rna 兼容, 并以核糖核酸蛋白格式交付, 这是实现更高特性的著名策略。

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