Improved vector toolkit for genome writing in mammalian cells

Area of Science:

• Synthetic Biology
• Genome Engineering
• Molecular Biology

Background:

• Efficient genome writing in mammalian cells necessitates robust methods for integrating large DNA payloads.
• The mammalian Switching Antibiotic resistance markers Progressively for Integration (mSwAP-In) method allows for iterative, biallelic genome rewriting with large DNA payloads (>100 kb).
• Previous limitations in mSwAP-In adoption were due to a lack of standardized vectors and technical constraints.

Purpose of the Study:

• To present an improved plasmid toolkit designed to streamline the implementation of the mSwAP-In method.
• To provide standardized and experimentally validated reagents for simplified large-scale genome writing.

Main Methods:

• Development of two core vectors: pLP-TK (landing-pad plasmid) and mSwAP-In MC2v2 (DNA assembly and delivery vector).
• Utilized Golden Gate and Gibson assembly methods for DNA integration.
• Incorporated various selection markers (positive, negative, fluorescent) and counterselection systems (FCU1/5-FC).
• Leveraged CRISPR/Cas9 for payload release and engineered vector architecture for propagation in yeast and E. coli.

Main Results:

• The toolkit streamlines mSwAP-In implementation for large DNA payloads (>100 kb) in mammalian stem cells.
• Characterized the FCU1/5-FC counterselection system in mouse embryonic stem cells, defining conditions to minimize bystander toxicity.
• Optimized Cas9-gRNA expression plasmids for common mSwAP-In applications.

Conclusions:

• The developed plasmid toolkit standardizes and simplifies large-scale genome writing using the mSwAP-In method.
• This resource facilitates broader adoption and application of advanced genome engineering techniques in mammalian cells.