Author ORCID Identifier
0000-0002-9263-1883
Date of Graduation
8-2024
Document Type
Dissertation (PhD)
Program Affiliation
Quantitative Sciences
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Glen Traver Hart
Committee Member
Scott Kopetz
Committee Member
Eduardo Vilar-Sanchez
Committee Member
John Paul Y.C. Shen
Committee Member
Linghua Wang
Abstract
The emergence of high-throughput sequencing technologies and the development of targeted cancer therapies have significantly advanced our understanding of cancer genomics and prolonged patient survival. Despite these advances, durable response remains difficult to achieve in the clinic. The concept of synthetic lethality has gained traction as a promising opportunity to discover novel cancer-specific vulnerabilities and therapeutic targets. Unfortunately, initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. In this dissertation, I report: 1) paralog selection method to select candidate synthetic lethal paralogs; 2) our Cas12a multiplex platform “IN4MER” that provides superior sensitivity and replicability to support genome- wide and genetic interaction screening; 3) observations and analyses to account for varied guide efficiency in multiplex CRISPR screen data.
Given the approximated 20,000 protein-coding genes in the human genome, the search space for synthetic lethal genes is too vast to be examined in a thorough “all by all” fashion. In the first part of this dissertation, I describe filtering method with which I narrowed the search space and obtained candidate synthetic lethal paralogs, triples and quadruples, all of which were subsequently tested utilizing our IN4MER platform.
In addition, synthetic lethality between two or more genes could not be examined in CRISPR Cas9-based single knockout libraries due to functional buffering. The Cas12a enzyme, unlike Cas9, can intrinsically process multiple guide RNAs from the CRISPR array, only requiring direct repeats (20 nucleotides), instead of tracrRNA (76 nucleotides) in Cas9 systems, thus freeing up space to include more guides in each array and supports multiplexing. In the second part of this dissertation, I describe our multiplex Cas12a IN4MER platform and screening results, targeting up to four genes from a single array. We constructed a genome- scale library and added candidate synthetic lethal paralogs, summing up to only 49k arrays, which is substantially smaller than a typical CRISPR/Cas9 monogenic library. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes, as well as the detection of synthetic lethal and masking/buffering genetic interactions, which are not currently supported by any other library. Importantly, the IN4MER platform offers a five-fold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for such combinatorial studies.
Lastly, I describe observations from investigating guide efficiencies in multiplex CRISPR screen data, as exemplified in KRAS guides, by conducting regression analysis and devising an alternative analysis method, the best FC method, to maximize hit discovery form screen data. The regression analysis demonstrated how sub-optimal guide efficiencies may hinder hit- calling, while results from the best FC method suggested that such occurrences are rare at the genome scale. Collectively, this work showcases the latest state-of-art CRISPR multiplex technology, and methods to utilize such technology to advance our understanding of synthetic lethality and functional genomics.
Keywords
CRISPR screen, synthetic lethality, functional genomics, genetic interaction