美国加州大学伯克利分校Jennifer A. Doudna团队近期取得重要工作进展，他们研究提出工程化改良的CRISPR-Cas9可加速DNA的解决促进基因编辑的效率。相关研究成果2024年5月22日在线发表于《细胞》杂志上。
据介绍，可恒温的簇状规则间距短回文重复序列（CRISPR）和CRISPR相关酶（Cas9）可以延长蛋白质的寿命，从而提高基因组编辑的效率和递送能力。
然而，初步实验表明，在培养的人类细胞中使用嗜热脂肪土芽孢杆菌Cas9（GeoCas9）时几乎没有活性。GeoCas9的实验室演化变体通过获得楔形（WED）结构域的突变克服了这一自然限制，其基因组编辑水平提高了100倍以上。野生型和改良型GeoCas9（iGeoCas9）酶的冷冻电镜（cryo-EM）结构显示，iGeoCas9的WED结构域与DNA底物之间存在扩展接触。
生化分析表明，iGeoCas9能在哺乳动物细胞（而非细菌细胞）典型的镁限制条件下加速DNA解旋以捕获底物。这些发现使得对其他Cas9同源物进行合理工程改造成为可能，从而提高了基因组编辑水平，为编辑酶的改良提供了一种通用策略。
总之，这一研究揭示了Cas9 WED结构域在DNA解旋中的新作用，并证明了加速目标解旋如何显著提高Cas9诱导的基因组编辑活性。
附：英文原文
Title: Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9
Author: Amy R. Eggers, Kai Chen, Katarzyna M. Soczek, Owen T. Tuck, Erin E. Doherty, Bryant Xu, Marena I. Trinidad, Brittney W. Thornton, Peter H. Yoon, Jennifer A. Doudna
Issue&Volume: 2024-05-22
Abstract: Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
DOI: 10.1016/j.cell.2024.04.031
Source: https://www.cell.com/cell/fulltext/S0092-8674(24)00457-4