Many of the cutting-edge innovations on the CRISPR/Cas9 system involve the use of complex sgRNA libraries. For example, such libraries have recently been used in functional genomics applications to screen for specific genes in both cell lines and living organisms, as well as in subcellular imaging to fluorescently label chromatin in living cells. However, chemically synthesizing customized libraries can be cost prohibitive, requires precise genomic information, and can takes several weeks. We developed a rapid and efficient enzymatic method to generate sgRNA libraries from arbitrary DNA substrates, significantly reducing the cost of library generation. A type IIS restriction enzyme binding site was incorporated directly into the sgRNA scaffold sequence without affecting the catalytic properties of the Cas9 complex, resulting in a simple method that generates high fidelity sgRNA libraries. Additionally, the library is constructed on the surface of streptavidin coated magnetic beads and a strand displacing polymerase is used to elute DNA from the beads, reducing the loss of material and simplifying purification between steps. We used this method to generate two sgRNA libraries. The first targets the entire E. coli genome, and the second targets genes expressed in the developing zebrafish heart. This second library will be used in a forward genetic screen in zebrafish to identify genes underlying heart defects. These complex sgRNA libraries show the utility of the method in generating libraries from DNA from any species, ranging from prokaryotes like bacteria, to eukaryotes including plants and animals.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-10282 |
| Date | 09 April 2019 |
| Creators | Yates, Joshua Don |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | https://lib.byu.edu/about/copyright/ |
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