Using high throughput CRISPR based methods for systematic evaluation of genome editing strategies in haemopoietic stem cells

Genome editing of haemopoietic cells is at the forefront of the clinical development the technology, with recent regulatory approval for Exagamglogene autoleucel for the treatment of haemoglobinopathies. Several variants of genome editing technologies have been developed over the last decade, which extend the capabilities of genome editing beyond that of the canonical nuclease-based proteins.

This involves inactivation of Cas9 nuclease function and the linkage of additional enzymes to Cas9 to allow direct targeting of specific DNA sequences. This has resulted in the development of base editors, which use deamination of nucleotides to make single base changes and prime editors, which use reverse transcriptase to enable an elongated guide RNA to be used as a template for DNA repair.

A further development are epigenome editors, which alter transcription through Cas9-targetted modification of DNA methylation and histone proteins but leave the DNA sequence unchanged.

All of these variants of genome editing have differing efficacy and result in variable sequence changes at specific sites in the genome. Furthermore, editing outcomes are highly variable between different cell types and it is common to find that high editing efficiencies in cell lines do not translate into primary cells. This is not fully understood but is likely due to cell type specific differences in the epigenetic landscape and DNA damage response.

This cell type variability has not been characterised systematically in key primary cell types such as HSCs. In this project we aim to develop methods of using high throughput CRISPR screens to systematically characterise the editing outcomes in haemopoietic stem cells. Once developed, we plan to compare editing outcomes with different editing modalities at different classes of genomic elements in different cell types.

This will potentially allow cell type specific prediction of editing strategies and inform on methods of manipulating the cellular program to enhance editing.