CRISPR/Cas9-mediated genome-editing of long-term hematopoietic stem cells

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K15872
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 52050:Embryonic medicine and pediatrics-related
• Research Institution: Jichi Medical University
• Principal Investigator: Byambaa Suvd 自治医科大学, 医学部, 客員研究員 (90834193)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: CRISPR/Cas9 / Hematopoietic stem cells / HDR / Genome-editing / beta-glucan / mTOR inhibitor / GSK inhibitor / Engraftment

Outline of Final Research Achievements

To improve in vitro HDR editing efficiency of expanded/edited long-term HSCs, we tested first, GSK and mTOR inhibitors which are known to support the maintenance and self-renewal of HSCs. Treatment with two inhibitors, HDR-mediated editing improved from ~5% to 10-30% in vitro; from ~1% to ~10% in vivo 2) Modified single-strand DNA template to conjugate the template to Cas9, and it showed 5-20% in vitro; ~5% in vivo after serial transplantation. Second, we established and optimized the method to improve the edited cell engraftment by using beta-glucan. Our result showed that beta-glucan protects HSCs and improves their engraftment ability. These results showed that ex vivo expanded and HDR-edited cells
are capable to repopulate HDR-edited multilineage cells after transplantation, demonstrating that precise genome editing of expanded long-term HSCs is feasible.

Free Research Field

Regenerative medicine

Academic Significance and Societal Importance of the Research Achievements

Genome-edited HSCs engraftment capability is an important point for successful HSCs transplantation. This work shows the ex vivo treatment of beta-glucan before the genome-editing process has protective effects for genome-edited HSCs to survive in the recipient body after transplantation.