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[Correspondence]Failure to detect DNA-guided genome editing using Natronobacterium gregoryi Argonaute

Seung Hwan Lee1,10, Giandomenico Turchiano2,3,10, Hirotaka Ata4,10, Somaira Nowsheen4, Marianna Romito2,3,5, Zhenkun Lou6, Seuk-Min Ryu1,7, Stephen C Ekker8, Toni Cathomen2,3,9 & Jin-Soo Kim1,7

Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, South Korea. 2Institute for Cell and Gene Therapy, Medical Center.University of Freiburg, Freiburg, Germany. 3Center for Chronic Immunodeficiency, Medical Center.University of Freiburg, Freiburg, Germany. 4Mayo Graduate School, Mayo Clinic, Rochester, Minnesota, USA. 5Graduate School, Faculty of Biology, University of Freiburg, Freiburg, Germany. 6Division of Oncology Research, Mayo Clinic, Rochester, Minnesota, USA. 7Department of Chemistry, Seoul National University, Seoul, South Korea. 8Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA. 9Faculty of Medicine, University of Freiburg, Freiburg, Germany. 10These authors contributed equally to this work.

Correspondence to : Toni Cathomen or Stephen C Ekker or Jin-Soo Kim

To the Editor:
DNA-guided DNA cleavage using Argonaute-family proteins was previously reported but of limited practical utility due to the requirement for supraphysiological conditions, such as high temperature1, 2. Recently, Gao et al.3 reported an Argonaute protein isolated from Natronobacterium gregoryi (NgAgo) as a genome engineering tool for editing the human genome. After transfecting human cell lines with a plasmid DNA encoding NgAgo and a 24-nucleotide, 5′-phosphorylated single-strand guide DNA (gDNA), they showed gene editing at endogenous targets. Here, we report the results of three different groups (Cathomen, Supplementary Fig. 1 and Supplementary Methods 1; Ekker, Supplementary Fig. 2 and Supplementary Methods 2; and Kim, Supplementary Fig. 3 and Supplementary Methods 3), independently attempting to reproduce the original findings of Gao et al.3, specifically focusing on the evidence for DNA edits in cultured human cell lines. All three groups synthesized the same 5′-phosphorylated gDNA sequences and used the NgAgo vector provided by Gao et al.3 via Addgene (Cambridge, MA, USA) to transfect the same cell lines, and analyzed the genomic DNA for signs of gene editing. Controls confirmed efficient delivery of both plasmid DNA and gDNAs as well as expression of the NgAgo protein. Despite various attempts to optimize NgAgo-mediated genome editing in three of the reported cell lines, no evidence of successful editing of endogenous target sequences was detected.

- 형식: Correspondence
- 게재일: 2017년 01월 (BRIC 등록일 2017-01-16)
- 연구진: 국내(교신)+국외 연구진태극기
- 분야: Biotechnology, Genetics
Dot/Icm 제 4 유형 커플링 단백질 복합체의 선택적 이펙터 단백질 인식[Nat. Commun.]
발표: 김현민 (KAIST)
일자: 2020년 9월 29일 (화) 오후 02시 (한국시간)
언어: 한국어
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