Engineered CRISPR-Cas enzymes with enhanced properties to enable novel genetic medicines

Abstract

Programmable genome targeting with CRISPR-Cas enzymes is a critical biological innovation underpinning biological research and a burgeoning class of transformative therapeutics. While the user can choose nearly any genetic sequence against which to program the guide RNA, in practice, all CRISPR enzymes have a major limitation constraining the availability of targetable genomic or transcriptomic sequences. In this dissertation, I explore varied research directions all relating to CRISPR-Cas target acquisition by first extensively characterizing the efficiency, safety, and potential impact of engineered SpCas9 variants capable of non-canonical PAM recognition, attempt to apply our knowledge of precision genomic positioning to create a novel CRISPR-based technology for proximity-dependent activation, and explore potentially extending engineering methodologies to RNA-targeting enzymes which we previously used in DNA-targeting modalities. For Cas9 enzymes, the inability to target any sequence arises due to the requirement to recognize a protospacer adjacent motif (PAM) to initiate target site binding. The necessity of PAM readout by Cas9 enzymes thus limits the feasibility of precisely and efficiently installing a wide range of edits, especially when using technologies that have an increased dependence on edit proximity, namely base editors. Engineered Cas9 variants with relaxed PAM recognition attempt to address this issue by enabling nearly nucleotide-level positioning of the Cas9 protein, but in some cases can result in increased off-targets and/or reduced on-target activity. To overcome these challenges and to enable efficient and specific genome editing, we developed a suite of Cas9 variants with altered PAM preferences to enable precise genomic positioning while reducing off-target edits. Our engineering strategy yielded a toolbox of novel Cas9 variants that efficiently produce insertion of deletion mutations (indels), C-to-T edits, and A-to-G edits on targets with non-NGG PAMs (as nucleases, CBEs, and ABEs, respectively). We then applied these enzymes for various pre-clinical applications such as base editors for the installation of protective genetic variants against a variety of serious diseases, model cell line correction of pathogenic mutations in sickle cell anemia and alpha-1 anti-trypsin deficiency, and correction of mutations in patient-derived primary cells. Together these enzymes were shown to enable precise editing of any target with an NGN PAM without many of the drawbacks of broadly targetable Cas9 variants such as SpRY, SpG, or others. I next detail my efforts towards the development of an original CRISPR-based technology that enact new biological effects based on proximity activation of fused enzymatic domains to the Cas protein. Cas proteins have proven to be a modular scaffold capable of consistent function even when fused to other enzymatic constructs (such as base editors, prime editors, and more), and I proposed to leverage this property to create a system capable of making a variety of biological signals sequence dependent. By using the Cas9 or Cas13 proteins as a programmable DNA-localization domain, I sought to activate via proximitydependent dimerization a variety of effectors such as cell-killing caspases, pro-drug metabolizing enzymes, and split-complementation luciferases. This work produced novel findings of how Cas protein localization can work in practice when applied to the effective concentration of these fused domains. Lastly, I briefly explored the potential to extend some of the engineering strategies implemented in DNA-targeting proteins to RNA-targeting CRISPR systems. While DNAtargeting Cas9 or Cas12, for example, require a PAM to initiate successful target acquisition, many RNA-targeting systems do not share this constraint and can target nearly any sequence. However, in practice, RNA knockdown efficiency can vary broadly based on the RNA secondary structure of the target. In seeking to engineer Cas13 systems, I sought to make them less dependent on accessible RNA structures and therefore more capable of localizing to any sequence in the transcriptome. In the course of this work, I, along with collaborators, also explored the interference of Cas13 target acquisition by putative anti-CRISPR proteins. This work resulted in a deeper understanding of the complexities of engineering RNA-targeting systems and the refutation of ineffective anti-CRISPR proteins reported by another group.