Author ORCID Identifier

Date of Graduation


Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Giulio Draetta

Committee Member

Andy Futreal

Committee Member

Richard Behringer

Committee Member

Traver Hart

Committee Member

Haoqiang Ying


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.


CRISPR, Functional genomics, Pooled library screening, CRISPR/Cas12a



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