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扩展基因组编辑工具箱与Abyssicoccus albus Cas9 使用一个独特的protospacer相邻的基因序列

Akiyoshi Nakamura¹, Hiroshi Yamamoto¹, Tsubasa Yano²

¹Bioproduction Research Institute, The National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

The CRISPR journal

|August 7, 2024

概括

研究人员发现了Abyssicoccus albus Cas9 (AalCas9),一个较小的CRISPR-Cas9酶,识别了一种新的5'-NNACR-3'PAM序列. 这扩展了植物和人类细胞的基因组编辑工具.

科学领域:

分子生物学分子生物学
遗传学遗传学是一个
生物技术是生物技术.

背景情况:

克里斯普尔-Cas9基因组编辑依赖于对DNA结合的原空间邻基因 (PAM) 识别.
广泛使用的Streptococcus pyogenes Cas9 (SpyCas9) 针对5'-NGG-3' PAM,限制其在不同基因组区域的应用.
工程SpyCas9和探索Cas9的正方体是扩大基因组编辑能力的关键策略.

研究的目的:

识别和描述具有明显PAM特异性的新型Cas9正义词.
为了评估植物和人类细胞中新发现的Cas9酶的基因组编辑效率.
调查PAM识别和工程师Cas9的结构基础,以改变序列偏好.

主要方法:

生物信息识别和特征 Abyssicoccus albus Cas9 (AalCas9) 的正统.
在体外和体内对AalCas9的5'-NNACR-3' PAM序列识别的实验验证.
导向RNA序列的优化,用于增强AalCas9介导的基因组编辑.
结构预测和位点定向的突变发生,以探测PAM识别机制.

主要成果:

一个较小的Cas9正义体,AalCas9,被确定,识别了一个独特的5'-NNACR-3'PAM序列.
修改导向RNA显著提高了AalCas9编辑效率,在植物和人类细胞系中.
假定PAM识别部位的点突变改变了AalCas9的序列偏好,由结构预测证实.

结论:

AalCas9代表了基因组编辑的新工具,扩大了可向的基因组站点的范围.
导向RNA和蛋白质工程的优化可以提高新的Cas9变体的实用性.
了解Cas9多样性为开发下一代基因组编辑技术提供了宝贵的见解.

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