没有可检测的全基因组非目标突变的体内CRISPR编辑
Pinar Akcakaya1, Maggie L Bobbin2,3,4, Jimmy A Guo2,3
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
Nature
|September 14, 2018
概括
一种新的方法,即体内非目标基因验证 (VIVO),可靠地在体内检测出CRISPR-Cas的非目标基因突变. VIVO 显示基因编辑在小鼠肝脏中是安全有效的.
科学领域:
- 生物技术
- 遗传学
- 分子生物学
背景情况:
- CRISPR-Cas基因组编辑具有治疗潜力,但需要识别非目标突变以获得临床安全性.
- 目前还没有有效的方法可靠地检测体内非目标突变,这阻碍了临床转换.
研究的目的:
- 引入一种高度敏感的策略,即在体内验证非目标基因 (VIVO),以便在体内稳定地识别CRISPR-Cas核酶的全基因组非目标效应.
- 在体内评估由CRISPR-Cas核酶诱导的非目标突变的频率和影响.
主要方法:
- 开发和应用体外目标 (VIVO) 验证策略.
- 使用一个有意识的导向RNA来测试VIVO在检测小鼠肝脏中的异位突变的灵敏度.
- 使用VIVO来评估适当设计的导向RNA用于体内编辑的安全性.
主要成果:
- 当使用杂交导向RNA时,CRISPR-Cas核酶可以在小鼠肝脏中诱导大量的异位突变.
- 适当设计的导向RNA可以在没有可检测的异位突变的小鼠肝脏中有效编辑.
- 证明VIVO是一种敏感且强大的全基因组外位分析方法.
结论:
- 维沃策略提供了一种可靠的方法来定义和量化整体生物中的基因编辑核酶的非目标效应.
- 谨慎的引导RNA设计对于实现安全有效的体内基因编辑至关重要.
- VIVO为开发使用体内基因编辑的更安全的治疗策略提供了蓝图.
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