Enhancing CRISPR/Cas9 Gene Editing of Human Hematopoietic Stem Cells

Review of “Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair” from STEM CELLS by Stuart P. Atkinson 

The DNA double-strand breaks generated by the CRISPR/Cas9 gene editing system can be repaired throughout the cell cycle by the error‐prone non-homologous end joining (NHEJ) pathway or during S/G2 phases by the more precise homology‐directed repair (HDR). The application of HDR‐mediated gene editing in human hematopoietic stem cells to precisely correct disease‐causing mutations suffers from a relatively low efficiency in primitive long‐term reconstituting stem cells [1-3], which currently prohibits the exploration of ex vivo correction of hematopoietic stem cells as a therapeutic option for a range of diseases/disorders.

In a new STEM CELLS study, researchers led by Donald B. Kohn (University of California Los Angeles, Los Angeles, California, USA) now report on their attempts to enhance HDR-associated gene editing in human hematopoietic stem cells. To this end, Lomova et al. now establish that the application of a Cas9 variant with decreased nuclease activity in G1 and M phases coupled with S/G2 cell synchronization can improve the efficiency of hematopoietic stem cell gene editing in vitro and in vivo [4].

To create a Cas9 variant that preferentially promotes HDR in the S/G2 phase of the cell cycle, the authors of the study followed a previously reported strategy [5] by fusing a fragment of the Geminin domain to the C‐terminus of Cas9 to allow the protein to be ubiquitinated and degraded by the APC/Cdh1 complex in G1 and late M phases. The study then employed a selective inhibitor of CDK1 to transiently arrests cells at the G2‐M transition for synchronization purposes. Encouragingly, the in vitro assessment of this enhanced gene-editing strategy provided evidence an improvement in the HDR/NHEJ ratio, a finding confirmed in later xenotransplantation studies of edited human hematopoietic stem cells in immunodeficient mice.

Overall, the application of cell cycle‐dependent control of nuclease activity and DNA repair pathways as a means to enhance gene-editing in hematopoietic stem cells represents a clinically-relevant strategic approach to improve treatments for congenital diseases of the blood system. 

Can we extend this approach to promote enhanced gene-editing in other stem cell types? Stay tuned to the Stem Cells Portal to find out!

References

1. Genovese P, Schiroli G, Escobar G, et al., Targeted genome editing in human repopulating haematopoietic stem cells. Nature 2014;510:235.
2. Hoban MD, Cost GJ, Mendel MC, et al., Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 2015;125:2597-2604.
3. Wang J, Exline CM, DeClercq JJ, et al., Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nature Biotechnology 2015;33:1256.
4. Lomova A, Clark DN, Campo-Fernandez B, et al., Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair. STEM CELLS 2019;37:284-294.
5. Gutschner T, Haemmerle M, Genovese G, et al., Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair. Cell Reports 2016;14:1555-1566.