Tools used for genetic engineering
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Tools Used for Genetic Engineering
CRISPR-Cas Systems in Genetic Engineering
CRISPR-Cas9 and CRISPR-Cas12a
The CRISPR-Cas systems, particularly CRISPR-Cas9 and CRISPR-Cas12a, have revolutionized genetic engineering by enabling precise gene editing. These systems utilize a guide RNA (gRNA) to direct the Cas nuclease to a specific DNA sequence, where it introduces double-strand breaks that can be repaired to create targeted mutations1 3. The CRISPR-Cas9 system has been optimized for various organisms, including Drosophila melanogaster, where it allows for efficient germline and somatic genome engineering3. Additionally, the CRISPR-Cas12k system has been developed to achieve high-efficiency genomic integration and transcriptional regulation in challenging strains, such as Sinorhizobium meliloti2.
Nuclease-Dead Cas Molecules (dCas)
Beyond gene editing, nuclease-dead Cas molecules (dCas9 and dCas12a) offer a platform for precise control of genome function without cutting the DNA. These dCas tools are used for transcription regulation, epigenetic engineering, genome imaging, genetic screens, and chromatin immunoprecipitation1. The versatility of dCas tools makes them invaluable for synthetic biology and genetic engineering applications.
TALENs and Other Nucleases
Transcription Activator-Like Effector Nucleases (TALENs)
TALENs are another powerful tool for genetic engineering, particularly in plants. They enable targeted modifications of monocot and dicot genomes and can be used in conjunction with CRISPR/Cas9 systems for more sophisticated genome edits. TALENs are systematized for fast, modular cloning and can create single or multiple gene knockouts and large chromosomal deletions4.
Cas9 Nickases
Cas9 nickases, which induce single-strand breaks instead of double-strand breaks, are used for precise gene targeting. When delivered on geminivirus replicons, Cas9 nickases achieve gene targeting frequencies comparable to native Cas9, enhancing the precision of genetic modifications4.
High-Throughput Genetic Engineering Tools
SynBioSuite
SynBioSuite is a cloud-based tool that automates the design, construction, editing, simulation, and sharing of genetic parts and circuits. It integrates various software tools like SBOLCanvas, iBioSim, and SynBioHub to streamline the genetic circuit design process, reducing manual errors and improving efficiency5.
Gene Designer
Gene Designer is a stand-alone software that facilitates the design of synthetic DNA segments. It allows users to add, edit, and combine genetic elements through an intuitive drag-and-drop interface. The software includes features for codon optimization, sequencing primer generation, and restriction site management, making it a comprehensive tool for synthetic biology7.
Specialized Toolkits for Specific Organisms
Vibrio fischeri Engineering Tools
For the model organism Vibrio fischeri, a set of tools has been developed to rapidly engineer deletions and insertions without cloning. These tools use PCR splicing by overlap extension (SOEing) with antibiotic cassettes, allowing for direct introduction of DNA products by natural transformation. This method simplifies the generation of deletion mutants and gene insertions, facilitating studies on symbiosis, quorum sensing, and bioluminescence6.
Saccharomyces cerevisiae Toolkit
A Cas9-based toolkit has been created for Saccharomyces cerevisiae, addressing common obstacles in metabolic engineering. This toolkit includes a variety of Cas9-sgRNA plasmids, promoters, and protein tags, along with a web-based tool for automating DNA fragment generation. It enables efficient chromosomal integration, promoter selection, and protein localization, significantly improving the speed and ease of genetic edits in yeast10.
Conclusion
The tools used for genetic engineering are diverse and continually evolving. CRISPR-Cas systems, TALENs, high-throughput software tools, and specialized toolkits for specific organisms all contribute to the advancement of genetic engineering. These tools enable precise modifications, efficient gene regulation, and streamlined workflows, paving the way for innovative applications in synthetic biology and beyond.
Sources and full results
Most relevant research papers on this topic
A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology.
CRISPR-dCas tools enable precise control of gene expression without gene editing, enabling applications in genetic engineering and synthetic biology.
Highly CitedA versatile Cas12k-based genetic engineering toolkit (C12KGET) for metabolic engineering in genetic manipulation-deprived strains
The Cas12k-based genetic engineering toolkit (C12KGET) efficiently integrates and regulates genes in challenging strains, enabling high-yield production of high-value-added chemicals in wild species.
Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila
Optimized CRISPR/Cas tools enable efficient germline and somatic genome engineering in Drosophila, enabling high-throughput genetic screening and revealing mutant phenotypes.
Highly CitedA Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants[OPEN]
Our toolkit enables targeted, specific genome editing in diverse plant species, enabling targeted gene knockouts and large chromosomal deletions.
Highly CitedSynBioSuite: A Tool for Improving the Workflow for Genetic Design and Modeling.
SynBioSuite improves the workflow for genetic design and modeling by automating the setup and reception of results for simulating designed circuits using an application programming interface.
Tools for Rapid Genetic Engineering of Vibrio fischeri
Our tools enable rapid genetic engineering of Vibrio fischeri, accelerating mechanistic studies of biological processes without cloning.
Rigorous JournalGene Designer: a synthetic biology tool for constructing artificial DNA segments
Gene Designer is a software tool that simplifies the design of synthetic DNA segments for molecular biologists, enabling efficient creation of functional genetic constructs for applications like codon optimization and protein engineering.
Highly CitedHigh-throughput genetic engineering tools for regulating gene expression in a microbial cell factory
High-throughput genetic engineering tools can optimize gene regulation in microbial cell factories, enhancing bioproduction performance and cellular diversification.
Tools and Principles for Microbial Gene Circuit Engineering.
Microbial gene circuit engineering is possible with versatile components and tools, enabling robust and scalable circuit performance and enabling rapid identification and correction of circuit failures.
Highly CitedA Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae
This CRISPR/Cas9 toolkit simplifies metabolic engineering in yeast, enabling 25-fold improvement in taxadiene production by optimizing enzyme expression.
Highly Cited