High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.
High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.
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DOI：
10.1038/s41587-022-01410-2
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发表时间：
2023-01
影响因子：
46.9
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Huang, Tony P.;Heins, Zachary J.;Miller, Shannon M.;Wong, Brandon G.;Balivada, Pallavi A.;Wang, Tina;Khalil, Ahmad S.;Liu, David R.
自然发生的和实验室创造的Cas9变体提供了一套Cas9蛋白，这些Cas9蛋白与多种原间隔器邻近基序（protospacer-邻基序，PAM）序列上的DNA靶点结合。进化和工程化的PAM变体已被证明对治疗性的体外和体内精确基因编辑至关重要。一些基因组位点，特别是那些富含嘧啶的PAM序列，仍然无法被高活性Cas9变体所访问。此外，工程广泛的PAM序列兼容性可以增加脱靶活性。利用噬菌体辅助非连续进化（PANCE）和evolver支持的噬菌体辅助连续进化（ePACE），我们将紧凑的Cas9变体Nme2Cas9进化为新的单核苷酸嘧啶PAM识别。我们开发了一个通用的选择策略，需要功能性编辑，同时允许目标原间隔器和PAM完全指定。我们应用这种选择来进化四种新的、高活性的Nme2Cas9变体。进化变种eNme2-C和eNme2-C。NR分别在含有N4CN pam的位点上进行有效的碱基编辑和核酸酶介导的indel形成，其中N可以是任何核苷酸。eNme2-T变体。1和eNme2-T。2 .在许多N4TN PAM序列上进行腺嘌呤碱基编辑。与SpRY相比，唯一报道的Cas蛋白变体能够参与类似范围的嘧啶PAMs， eNme2-T。1和eNme2-T。2以相当的效率提供对N4TN PAM序列的替代访问，而eNme2-C和eNme2-C。NR提供较少的限制性PAM要求，在各种人类细胞类型中具有相当或更高的活性，并且在N4CN PAM序列上具有更低的脱靶活性。总之，这些进化的Nme2Cas9变体能够靶向大多数富含嘧啶的PAM序列，包括那些现有Cas蛋白难以进入的序列，从而大大扩展了基于cas9的技术的靶向能力。
Naturally occurring and laboratory-created Cas9 variants have provided a suite of Cas9 proteins that engage DNA targets at a variety of protospacer-adjacent motif (PAM) sequences. Evolved and engineered PAM variants have proven critical to therapeutic ex vivo and in vivo precision gene editing. Some genomic loci—especially those with pyrimidine-rich PAM sequences—remain inaccessible by high-activity Cas9 variants. Moreover, engineering broad PAM sequence compatibility can increase off-target activity. Using phage-assisted non-continuous evolution (PANCE) and eVOLVER-supported phage-assisted continuous evolution (ePACE), we evolved Nme2Cas9, a compact Cas9 variant, towards novel, single-nucleotide pyrimidine PAM recognition. We developed a general selection strategy that requires functional editing while allowing the target protospacer and PAM to be fully specified. We applied this selection to evolve four new, high-activity Nme2Cas9 variants. Evolved variants eNme2-C and eNme2-C.NR enable efficient base editing and nuclease-mediated indel formation, respectively, at sites containing N4CN PAMs, where N can be any nucleotide. Variants eNme2-T.1 and eNme2-T.2 enable adenine base editing at many N4TN PAM sequences. When compared to SpRY, the only reported Cas protein variant capable of engaging a similar range of pyrimidine PAMs, eNme2-T.1 and eNme2-T.2 offer alternative access to N4TN PAM sequences at comparable efficiencies, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types, and much lower off-target activity at N4CN PAM sequences. Together, these evolved Nme2Cas9 variants enable targeting of most pyrimidine-rich PAM sequences, including those poorly accessed by existing Cas proteins, substantially expanding the targeting capabilities of Cas9-based technologies.
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影响因子：
46.9
作者：
Huang, Tony P.;Heins, Zachary J.;Miller, Shannon M.;Wong, Brandon G.;Balivada, Pallavi A.;Wang, Tina;Khalil, Ahmad S.;Liu, David R.
通讯作者：
Liu, David R.
影响因子：
46.9
作者：
Kim YB;Komor AC;Levy JM;Packer MS;Zhao KT;Liu DR
通讯作者：
Liu DR
影响因子：
16.6
作者：
Badran AH;Liu DR
通讯作者：
Liu DR
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
48
作者：
DeBenedictis, Erika A.;Chory, Emma J.;Esvelt, Kevin M.
通讯作者：
Esvelt, Kevin M.