ArticleScientific reports2019
Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9.
Article in Scientific reports, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 77 citations in OpenAlex.
- Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases.Nature biomedical engineering · 2025Article
- Progress and Prospects of Gene Editing in Pluripotent Stem Cells.Biomedicines · 2023Review
- Visual function restoration in a mouse model of Leber congenital amaurosis via therapeutic base editing.Molecular therapy. Nucleic acids · 2023Article
- Precise DNA cleavage using CRISPR-SpRYgests.Nature biotechnology · 2023Article
- Off-target effects of base editors: what we know and how we can reduce it.Current genetics · 2022Review
- Adenine base editor engineering reduces editing of bystander cytosines.Nature biotechnology · 2021Article
- High-purity production and precise editing of DNA base editing ribonucleoproteins.Science advances · 2021Article
- Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment.Nature protocols · 2021Article
- CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral integration sites.Molecular therapy. Methods & clinical development · 2021Article
- Base and Prime Editing Technologies for Blood Disorders.Frontiers in genome editing · 2021Review
- Article
- A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells.BMC biology · 2020Article
- Current Status and Challenges of DNA Base Editing Tools.Molecular therapy : the journal of the American Society of Gene Therapy · 2020Review
- CGD: Comprehensive guide designer for CRISPR-Cas systems.Computational and structural biotechnology journal · 2020Article
- Revisiting CRISPR/Cas-mediated crop improvement: Special focus on nutrition.Journal of biosciences · 2020Article
- An adenine base editor with expanded targeting scope using SpCas9-NGv1 in rice.Plant biotechnology journal · 2019Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.