Precise and expansive genomic positioning for CRISPR edits

• Main Author: Jakimo, Noah Michael.
• Other Authors: Joseph M. Jacobson.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2020
• Subjects: Program in Media Arts and Sciences
• Online Access: https://hdl.handle.net/1721.1/123626

Thesis: Ph. D., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2019 === Cataloged from PDF version of thesis. === Includes bibliographical references (pages 91-105). === The recent harnessing of microbial adaptive immune systems, known as CRISPR, has enabled genome-wide engineering across all domains of life. A new generation of gene-editing tools has been fashioned from the natural DNA/RNA-targeting ability of certain CRISPR-associated (Cas) proteins and their guide RNA, which work together to recognize and defend against infectious genetic threats. This straight-forward RNA-programmed sequence recognition by CRISPR has facilitated its rapid global impact on genetic research, diagnostics, therapeutics, and bioproduction. An ideal DNA-editing platform would achieve perfect accuracy on any desired cellular and genomic target. CRISPR systems, however, have limited target fidelity and range, in part due to their evolutionary pressures to defend microbes from fast-mutating viruses without self-targeting their own guide RNA. === These natural limitations of CRISPR can especially constrain gene-editing in animals and plants, which are more vulnerable to off-target activity occurring in one of their trillions of cells with genomes that are 1000x larger than those of unicellular microbes that natively harbor CRISPR systems. This thesis overcomes three critical challenges for precise and broad gene-editing of complex organisms: 1) engineering a means of specificity for the type of cells to edit, 2) improving target-matching accuracy, and 3) broadening the editable portion of the genome. === This thesis addresses these challenges by integrating custom developed computational design tools and biological validation of the resulting novel CRISPR systems; 1) To target within multicellular heterogeneity, new oligonucleotide-sensing structural motifs are designed and embed into guides that can potentially control CRISPR nuclease activity based on cell-type transcriptome patterns; 2) To discern among increased similarity between a target and non-target sequences in larger genomes, base-pairing thermostability principles are employed to tune the biochemical composition of guides that can evade subtly mismatched off-target sites; 3) To expand the reach of editing techniques with narrow windows of operation, such as base-editing, bioinformatics workflows that discover previously uncharacterized Cas proteins with novel target scope are created. === This thesis demonstrates the effectiveness of these strategies in the context of in vitro, bacterial, and human cell culture assays, and contributes advancements in the precision and generality for CRISPR gene-editing. === by Noah Michael Jakimo. === Ph. D. === Ph.D. Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences