Multiplexed Crispr Libraries For Cancer Functional Genomics

<p>High-throughput forward genetic screenings are invaluable tools to systematically explore genetic interactions and to link gene disruption with disease contexts. The adaptation of CRISPR/Cas9 has improved the sensitivity and specificity of functional screenings. Despite this advance, there remains a long-standing need to improve functional screenings with smaller and more versatile pooled libraries. Capitalizing on the inherent multiplexing capability of a class 2 CRISPR enzyme AsCpf1, we developed a multiplexed, high throughput screening strategy that has avoided the usual trade-off between library size and library penetration, allowing library minimization without sacrificing gene targeting efficiency. We optimized the AsCpf1 protein for functional genomics use and demonstrated that an AsCpf1-based multiplexed library outperforms its monocistronic CRISPR/Cas9 library counterpart with a greatly reduced library size. With this strategy, we constructed the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n=17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2020

Authors dc:creator

• Liu, Jintan
• <p>https://orcid.org/0000-0001-6609-9777</p>

Contributors dc:contributor

• Giulio Draetta
• Andy Futreal
• Richard Behringer

• CRISPR
• Functional genomics
• Pooled library screening
• CRISPR/Cas12a
• Biotechnology
• Genomics
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/995

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-2045