Gene Editing With Crispr-Cas9 For Treating Beta-Hemoglobinopathies

CRISPR/Cas9 systems provide a powerful tool for precision genome editing due to their highly efficient targeting of specific DNA sequences in a genome. We have designed CRISPR single-guide RNAs (sgRNAs) containing the guide and tracr RNAs to selectively edit the human globin locus for potential therapeutic benefit in treating sickle cell anemia and beta-thalassemia. Multiple sgRNAs have been identified and tested across key sites in the human globin locus, and the gene modification frequencies quantified in 293T, K562 and CD34+ hematopoietic progenitor cells. The nuclease cutting efficiency was determined by the rate of insertions, deletions or mutations (indels) from NHEJ-directed repair. The indel rates for these sgRNAs ranged from u003c1% to ~70%. We found that there was only a weak correlation between indel rates and deletion frequencies, and the gene modification efficiency varied between cell lines/cell types. Although bioinformatics tools are available for sgRNA design, determining the optimal sgRNAs is still largely empiric and system-dependent, thus better bioinformatics tools are needed. Our results demonstrate that, with careful design and optimization of sgRNAs, efficient genome editing can be achieved with high specificity, which promises effective treatment of beta-hemoglobinopathies.Disclosures Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership. Cornu: CRISPR Therapeutics: Research Funding. Miccio: CRISPR Therapeutics: Research Funding. Cradick: CRISPR Therapeutics: Employment. Cathomen: CRISPR Therapeutics: Consultancy, Research Funding. Lundberg: CRISPR Therapeutics: Employment. Mavilio: CRISPR Therapeutics: Consultancy, Research Funding.