Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs

• 成果类型: Article
• 署名作者: Le, Trang Thi; Choi, Hong Il; Kim, Ji Won; Yun, Jin - Ho; Lee, Yoon Hyeok; Jeon, Eun Jung; Kwon, Kil Koang; Cho, Dae - Hyun; Choi, Dong - Yun; Park, Su - Bin; Yoon, Hyang Ran; Lee, Jeongmi; Sim, Eun Jeong; Lee, Yong Jae; Kim, Hee - Sik
• 署名单位: Korea Research Institute of Bioscience & Biotechnology (KRIBB); University of Science & Technology (UST); Sungkyunkwan University (SKKU); Korea Research Institute of Bioscience & Biotechnology (KRIBB); Korea Research Institute of Bioscience & Biotechnology (KRIBB); Korea Research Institute of Bioscience & Biotechnology (KRIBB); University of Science & Technology (UST)
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12949
• DOI: 10.1073/pnas.2415072122
• 发表日期: 2025-03-03
• 关键词: nuclear-localization signal; chlamydomonas-reinhardtii; mediated transformation; crystal-structure; structural basis; protein; expression; transport; vird2

摘要

Pathogen-derived nuclear localization signals (NLSs) enable vigorous nuclear invasion in the host by the virulence proteins harboring them. Herein, inspired by the robust nuclear import mechanism, we show that NLSs originating from the plant infection-associated Agrobacterium proteins VirD2 and VirE2 can be incorporated into the Cas9 system as efficient nuclear delivery enhancers, thereby improving the low gene-editing frequency in a model microalga, Chlamydomonas reinhardtii, caused by poor nuclear localization of the bulky nuclease. Prior to evaluation of the NLSs, IPA1 (Cre04.g215850) was first defined in the alga as the nuclear import-related importin alpha (Imp alpha) that serves as a counterpart adaptor protein of the NLSs, based on extensive in silico analyses considering the protein's sequence, tertiary folding behavior, and structural basis when interacting with a well-studied SV40TAg NLS. Through precursive affinity explorations, we reproducibly found that the NLSs mediated the binding between the Cas9 and Imp alpha with nM affinities and visually confirmed that the fusion of the NLSs strictly localized the peptide-bearing cargoes in the microalgal nucleus without compensating for their cleavage ability. When employed in a real-world application, the VirD2 NLS increases the mutation frequency (similar to 1.12 x 10(-5)) over 2.4-fold compared to an archetypal SV40TAg NLS (similar to 0.46 x 10(-5)) when fused with Cas9. We demonstrate the cross-species versatility of the Imp alpha-dependent strategy by successfully applying it to an industrial into increasing the frequency of gene editing, which can be advantageously used in programmable mutagenesis with broad applicability.