Abstract: SA-PO450
Rapid and Efficient Method to Generate Donor Vectors for Homology-Directed Repair Mediated Genome Editing
Session Information
- Development and Regenerative Medicine
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Ester, Lioba, University of Cologne, Cologne, Germany
- Mccracken, Kyle, Boston Children''s Hospital, Boston, Massachusetts, United States
- Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background
Stem cell-derived kidney organoids are a powerful, tractable tool with numerous potential applications, including the investigation of complex processes underlying human kidney development. To further develop organoids as an embryologic model, it will be essential to generate genetic tools such as fluorescent reporters and lineage tracing systems, which have proven instrumental in rodent studies. With the emergence of CRISPR/Cas9 gene editing via homology directed repair (HDR) is becoming more common in pluripotent stem cells. However, construction of donor vectors that contain gene-specific homology regions remains a time-consuming and often costly endeavor. Thus, we simplified this process through the creation of a modular system to allow rapid and efficient cloning of homology arms into HDR donor vectors.
Methods
We designed and synthesized a plasmid with numerous features and cassettes that are easily interchangeable. These include fluorescent reporters, antibiotic resistance genes and an HSV-TK for use in negative selection. The genes for selection were contained in a floxed cassette to allow easy cre-mediated removal following successful integration. Gene-specific homology arms were created using high fidelity PCR, and final donor vectors were assembled using HiFi Cloning.
Results
To facilitate simultaneous dual and triple knock-in alleles, we used restriction cloning to generate a repertoire of vectors that pair different combinations of reporter and antibiotic resistance genes. The PCR primers designed to amplify the homology arms from genomic DNA of the cell lines of interest also contained sequences of overlap with the sites of insertion on the vector. Thus, in one step using HiFi cloning, we were able to assemble both homology arms into the double-digested vector. This process, including PCR of homology arms, cloning, and sequence validation, can be completed within one week at minimal cost.
Conclusion
Through creation of this vector collection and application of HiFi cloning, we have successfully devised an efficient, streamlined process for cloning donor vectors for use in HDR-mediated gene editing. Given the modular design, these plasmids can also be adapted to permit knock-in of any cDNA of interest. This approach is now broadly applicable for editing of any cell line, including human pluripotent stem cells and hPSC-derived kidney organoids.
Funding
- NIDDK Support