ArticleNucleic acids research2017
The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.
Article in Nucleic acids research, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 47 papers.
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Who cites it
47 citing papers in PubMed.
- Advances in large DNA fragment assembly for microbial cell factory engineering.Quantitative biology (Beijing, China) · 2026Review
- Strategies and mechanisms of precision genome engineering: From gene editing to genome writing.iMetaOmics · 2026Review
- Improving Gene Knock-In Efficiencies in Sheep Primary Cells Using a CRISPR/Cas9-Gal4 System.Current issues in molecular biology · 2025Article
- CRISPR-based shuttle cloning of 1397 human genes into UAS vectors.Acta biochimica et biophysica Sinica · 2025Article
- Biology and applications of CRISPR-Cas12 and transposon-associated homologs.Nature biotechnology · 2024Review
- How Helpful May Be a CRISPR/Cas-Based System for Food Traceability?Foods (Basel, Switzerland) · 2024Review
- CRISPR-Cpf1 system and its applications in animal genome editing.Molecular genetics and genomics : MGG · 2024Review
- Applications of Programmable Endonucleases in Sequence- and Ligation-Independent Seamless DNA Assembly.Biomolecules · 2023Review
- Optimization of CRISPR-Cas system for clinical cancer therapy.Bioengineering & translational medicine · 2023Review
- Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.Nucleic acids research · 2022Article
- A framework to efficiently describe and share reproducible DNA materials and construction protocols.Nature communications · 2022Article
- Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach.Nucleic acids research · 2022Article
- ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing.Nucleic acids research · 2021Article
- Programmed genome editing by a miniature CRISPR-Cas12f nuclease.Nature chemical biology · 2021Article
- Precise Characterization ofInsects · 2021Article
- CRISPAltRations: a validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing.Molecular therapy. Methods & clinical development · 2021Article
- Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.Nature communications · 2021Article
- Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters.Frontiers in bioengineering and biotechnology · 2021Review
- Strategies for mitochondrial gene editing.Computational and structural biotechnology journal · 2021Review
- Expanding application of CRISPR-Cas9 system in microorganisms.Synthetic and systems biotechnology · 2020Article
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6 authors.
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Abstract
As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.
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