ArticleNucleic acids research2016
CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.
Article in Nucleic acids research, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed, 74 citations in OpenAlex.
- Yeast nuclei-mediated precise delivery of synthetic megabase-scale human DNA into mammalian embryos.Nature protocols · 2026Review
- Engineering yeast chromosomal telomeres with a bacteriophage system.Nature communications · 2026Article
- Automated linear DNA assembly of A. thaliana's chloroplast and mitochondrial genome.Nucleic acids research · 2026Article
- Synthetic rewriting technologies in mammalian cells.Nature communications · 2026Review
- Assembly and delivery of large DNA via chromosome elimination in yeast.Nature protocols · 2025Review
- The design and engineering of synthetic genomes.Nature reviews. Genetics · 2025Review
- The conjugation-associated linear-BAC iterative assembling (CALBIA) method for cloning 2.1-Mb human chromosomal DNAs in bacteria.Cell research · 2025Article
- Recent technology advancements in large-scale DNA assembly.Quantitative biology (Beijing, China) · 2025Article
- A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster.Microorganisms · 2024Article
- Genome engineering on size reduction and complexity simplification: A review.Journal of advanced research · 2024Review
- CRISPR in Nucleic Acids Research: the sequel.Nucleic acids research · 2024Article
- "Cell Disk" DNA Storage System Capable of Random Reading and Rewriting.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Convenient synthesis and delivery of a megabase-scale designer accessory chromosome empower biosynthetic capacity.Cell research · 2024Article
- Automated high-throughput DNA synthesis and assembly.Heliyon · 2024Review
- Application and Technical Challenges in Design, Cloning, and Transfer of Large DNA.Bioengineering (Basel, Switzerland) · 2023Review
- YLC-assembly: large DNA assembly via yeast life cycle.Nucleic acids research · 2023Article
- Cross-species microbial genome transfer: a Review.Frontiers in bioengineering and biotechnology · 2023Review
- The TelN/tos-assisted precise targeting of chromosome segments (TAPE).Journal of advanced research · 2022Article
- Exogenous artificial DNA forms chromatin structure with active transcription in yeast.Science China. Life sciences · 2022Article
- Synthetic Genomics From a Yeast Perspective.Frontiers in bioengineering and biotechnology · 2022Review
Corrections and comments
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Authors and funding
4 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.
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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.