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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
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Developing a Cas9-based tool to engineer native plasmids in Synechocystis sp. PCC 6803.
Yi Xiao1,2, Shaojie Wang1, Sarah Rommelfanger3
1Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, Missori.
Biotechnology and Bioengineering
|June 14, 2018
Summary
This study introduces a novel Cas9 tool for rapid, one-step genome engineering in Synechocystis sp. PCC 6803 (S6803). The tool also enables efficient deletion of native plasmids and stable heterologous gene expression, advancing cyanobacterial biotechnology.
Area of Science:
- Microbiology
- Synthetic Biology
- Genetics
Background:
- Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium crucial for research and biotechnology.
- Its polyploidy complicates genome engineering, requiring inefficient multi-step selection processes.
Purpose of the Study:
- To develop a Cas9-based tool for one-step, segregation-free genome engineering in S6803.
- To create tools for efficient native plasmid deletion and stable heterologous gene expression in S6803.
Main Methods:
- Engineered a Cas9 tool for one-step genome modification.
- Utilized the Cas9 tool to delete native plasmids in S6803.
- Constructed a shuttle vector based on the native plasmid pCC5.2 for heterologous gene expression.
Main Results:
- Achieved one-step, segregation-free genome engineering in S6803.
- Successfully deleted three small native plasmids using the Cas9 tool.
- Demonstrated stable maintenance and functionality of heterologous genes on the shuttle vector for over 30 days without antibiotic pressure.
Conclusions:
- The developed Cas9 tool significantly improves the efficiency of S6803 genome engineering.
- The new tools facilitate rapid modification and stable expression of genes in S6803, benefiting fundamental research and biotechnological applications.
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