1 PhD project offered in the IPP winter call 2023/2024

Scientific Background

CRISPR has revolutionized both basic and applied biomedical research as an exceptional tool for programmable, targeted and precise genome editing. The DNA-targeting specificity of the CRISPR-associated endonuclease Cas9 has been enhanced by redesigning guide RNAs (gRNAs) and engineering variants with higher fidelity. However, control of Cas9 template-free editing in eukaryotic cells remains suboptimal for high precision therapeutic applications.

The repair outcome (the insertions and deletions, indels, associated with repair) of Cas9-induced DNA double strand breaks (DSBs) is not random but depends strongly on the sequence context of the target site, the type of DSB produced by Cas9 and can be modulated by mutations in Cas9 itself. The Roukos group recently developed novel Next-Generation Sequencing (NGS)-based methods to characterize alternative Cas9 cleavage patterns and established that certain types of Cas9 cuts are repaired by the cellular machinery to produce templated and predictable editing outcomes (Longo et al., submitted). The frequency of certain types of Cas9 induced DSBs varied greatly between target sites, and could be further modulated by mutations in Cas9 (Longo et al., submitted). These findings establish a clear mechanistic link between the type of DSB generate by Cas9 and repair outcome, and provide a unique opportunity for optimization of Cas9 towards predictable and precise gene editing, forming the basis of this PhD project.

PhD project: Engineering CRISPR-associated endonucleases for precise and predictable therapeutic genome editing

This project aims to engineer Cas9 variants that generate exclusively certain types of DSBs, allowing a predictable repair outcomes independently of the target sequence. For that we will establish fluorescent reporters for desirable repair outcomes in both yeast and human cells. We will use these reporters to perform targeted deep mutational scanning and random mutagenesis of Cas9 to identify Cas9 mutations with increased rates of desired outcomes and examine applications of such Cas9 variants for precise genome editing, including generation of gene knockouts and correction of disease variants in patient cells. The project combines work with both yeast and mammalian cells, and training on molecular, cellular and NGS-based methodologies. It combines the interests and expertise of the Roukos group in CRISPR biology and the development of genome-wide methods profiling Cas9 DNA incisions, the expertise of the Khmelinskii group in high-throughput screening and deep mutational scanning approaches, and the mutual interests in genome engineering.

If you are interested in this project, please select Khmelinskii/Roukos as your group preference in the IPP application platform.

Publications relevant to the project

Longo GMC, Sayols S, Mockel M, Beli P and Roukos V. (2023) Determinants of CRISPR/Cas9 Scissile Profile for Precise and Predictable Genome Editing, submitted

Schmid-Burgk JL, Gao L, Li D, Gardner Z, Strecker J, Lash B and Zhang F. (2020) Highly parallel profiling of Cas9 variant specificity, Mol Cell 78:794-800.e8 Link

Fowler D.M. and Fields S. (2014) Deep mutational scanning: a new style of protein science, Nat Methods 11:801-807 Link