김희식 (한국생명공학연구원, UST) | 한빛사논문 > 한빛사

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한국생명공학연구원, UST

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Proc. Natl. Acad. Sci. USA, Mar 11 2025, 122 (10) | https://doi.org/10.1073/pnas.2415072122 Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs

Trang Thi Le ^{1 2 †}, Hong Il Choi ^{1 2 †}, Ji Won Kim^{1 2 †}, Jin-Ho Yun^{1 2 3}, Yoon Hyeok Lee ⁴, Eun Jung Jeon ⁵, Kil Koang Kwon ⁵, Dae-Hyun Cho ¹, Dong-Yun Choi¹, Su-Bin Park ¹, Hyang Ran Yoon ⁶, Jeongmi Lee ^{7 8}, Eun Jeong Sim ^{1 2}, Yong Jae Lee ^{1 2 *}, Hee-Sik Kim ^{1 2 *}

¹Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, South Korea.
²Department of Environmental Biotechnology, University of Science and Technology, Daejeon 34113, South Korea.
³Department of Integrative Biotechnology, Sungkyunkwan University, Suwon-si, Gyeonggi-do 16419, South Korea.
⁴Design AI Lab, AI Center Samsung Electronics, Suwon-si, Gyeonggi-do 16678, South Korea.
⁵Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, South Korea.
⁶Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, South Korea.
⁷Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, South Korea.
⁸Department of Bio-Molecular Science, University of Science and Technology, Daejeon 34113, South Korea.

^†These authors contributed equally
^*Corresponding authors: correspondence to Yong Jae Lee or Hee-Sik Kim

Abstract

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 (Impα) 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 Impα 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 (~1.12 × 10^-5) over 2.4-fold compared to an archetypal SV40TAg NLS (~0.46 × 10^-5) when fused with Cas9. We demonstrate the cross-species versatility of the Impα-dependent strategy by successfully applying it to an industrial alga, Chlorella Sp. HS2. This work, focused on affinity augmentation, provides insights into increasing the frequency of gene editing, which can be advantageously used in programmable mutagenesis with broad applicability.

논문정보

형식Research article
게재일2025년 03월 (BRIC 등록일 2025-03-11)
연구진국내 연구진
분야 생명과학 > 생물공학

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