美国哈佛大学和麻省理工学院布罗德研究所David R. Liu和波士顿大学Ahmad S. Khalil课题组，揭示了单核苷酸嘧啶间隔区相邻基序(PAM)s紧凑型Cas9变体的高通量持续进化。2022年9月8日出版的《自然-生物技术》发表了这项成果。

通过定向进化，研究人员得到了四种Cas9变体，它们都可以靶向人类基因组中大多数富含嘧啶的PAM序列。使用噬菌体辅助非连续进化和eVOLVER噬菌体辅助连续进化，研究人员将紧凑型Cas9变体Nme2Cas9进化为可识别单核苷酸嘧啶-PAM序列的变体。研究人员研发了一种通用选择策略，该策略需要使用特定的protospacers和PAM进行功能编辑。

研究人员应用这种选择来进化高活性变体eNme2-T.1、eNme2-T.2、eNme2-C和eNme2-C.NR。变体eNme2-T.1和eNme2-T.2可实现对N4TN PAM序列的识别，具有与现有变体相当的编辑效率，而eNme2-C和eNme2-C.NR对PAM序列的要求较少，可对具有各种N4CN PAM序列的人类细胞进行识别并具有较低的脱靶活性。

据介绍，尽管存在具有不同原型PAM相容性的Cas9变体，但一些基因组位点，尤其是那些富含嘧啶PAM序列的基因组位点，仍然无法被高活性Cas9蛋白识别。此外，通过工程扩大PAM序列兼容性可以增加脱靶活性。

附：英文原文

Title: High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs

Author: Huang, Tony P., Heins, Zachary J., Miller, Shannon M., Wong, Brandon G., Balivada, Pallavi A., Wang, Tina, Khalil, Ahmad S., Liu, David R.

Issue&Volume: 2022-09-08

Abstract: Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci—especially those with pyrimidine-rich PAM sequences—remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to N4TN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at N4CN PAM sequences.

DOI: 10.1038/s41587-022-01410-2

Source: https://www.nature.com/articles/s41587-022-01410-2