Abstract:
Microarray DNA synthesis has revolutionized functional genomics, enabling the economical production of vast oligonucleotide libraries for genetic screens like RNAi and CRISPR-Cas9. Traditionally, the methods are restricted to cell culture, limiting the exploration of complex biological processes occurring in multicellular organisms, such as development, aging, and behavior. Additionally, bulk implementation of pooled libraries confines screening to an "all or nothing," often genome-wide, application. This necessitates the simultaneous administration of all oligonucleotides in a given library, limiting their application to varying experimental conditions.
This dissertation introduces a new set of tools and methods that enhance genetic engineering in Caenorhabditis elegans. Leveraging low-complexity libraries, I constructed CRISPR-Cas9 platforms with 189,390 target-specific oligonucleotides for either DNA- or RNP-based genome editing by creating compact gRNA designs (113 - 123 bp) addressable in an arrayed format through orthogonal dial-out PCR. The platforms support HDR- and NHEJ-mediated gene editing with adaptable experimental scales enabled by selective PCR-based retrieval of oligonucleotides containing gRNAs and homology arms. I utilized individual oligonucleotides to make large insertions (up to 7.1 kb) to tag and trap endogenous genes, exploring AlphaFold2-guided placements of fluorophores. Furthermore, I employed gRNAs that span gene loci to create PCR-detectable deletions in seven-transmembrane G protein-coupled receptors and to produce diverse genetic variants of the lipid-binding chaperone unc-119, generating a complex allele that confers temperature sensitivity. 
Although limitations, including double-stranded DNA toxicity, were observed in generating multiplexed edits, and further refinements are required to bring in vivo genomic imaging and somatic editing, the technology stands as a foundational platform. The platform's compatibility with expanding CRISPR-based applications and its applicability to other multicellular systems signal a broader shift toward studying and engineering biology at the organismal level. These affordable, scalable, and modular community resources bolster technical capabilities with advances in high-throughput sequencing and computational models, promoting a deeper understanding of complex biological traits and nuanced genetic studies.

Bio:
Mohammed D. Aljohani is pursuing his Ph.D. in Bioengineering at KAUST under the guidance of Associate Professor Christian Frøkjær-Jensen in the Laboratory of Synthetic Genome Biology. His research has advanced the use of synthetic chromosomes in C. elegans, particularly in enhancing transgenic techniques and exploring gene regulation in the germline. Mohammed’s efforts have led to the development of CRISPR-Cas9 platforms and the establishment of transgene designs that minimize silencing in repressive tissues, providing comprehensive transgenesis resources to the global scientific community.