在PAM相邻的DNA灵活性调中,CRISPR-Cas12a的目标外结合
Aleique Allen1, Brendon H Cooper2,3, Jaideep Singh1
1Department of Chemistry, University of Southern California, 3430 S Vermont Ave., Los Angeles, CA, 90089, USA.
Scientific reports
|February 10, 2025
概括
在原体空间邻基因 (PAM) 附近的DNA灵活性会影响Cas12a核酶的目标外结合. 在PAM+1,+2和+3位置的脱对增加了Cas12a的目标外活动,影响了基因组编辑应用程序.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物化学 生物化学
背景情况:
- Cas12a是一种用于基因组操纵的CRISPR相关核酶.
- 在Cas12a目标识别中,与原空间邻基因 (PAM) 邻的DNA灵活性是相关的.
研究的目的:
- 调查PAM相邻DNA灵活性和Cas12a目标外结合之间的关系.
- 了解PAM附近的DNA序列变异如何影响Cas12a的特异性.
主要方法:
- 调整了一种SELEX-seq方法来分析PAM+1到+6位置的DNA-DNA不匹配.
- 使用FnCas12a进行了体外结合测试,使用DNA库进行了不匹配的测试.
- 结合和不结合的DNA群体的测序确定了非目标结合倾向.
主要成果:
- 卡斯12a的非目标结合取决于PAM相邻的DNA灵活性.
- 需要在PAM+1的protospacer脱对,才能实现目标外的结合.
- 在PAM+2和+3位置脱配时,目标外结合会增加.
结论:
- 与PAM相邻的DNA灵活性可以调节Cas12a的目标外结合.
- DNA 的物理性质影响了Cas12a 的目标歧视.
- 这些发现对优化基于Cas12a的基因组编辑工具有意义.
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