A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Christopher A Vakulskas, Integrated DNA Technologies, Inc., Coralville, IA, USA.
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  • Daniel P Dever, Department of Pediatrics, Stanford University, Stanford, California, USA.
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  • Garrett R Rettig, Integrated DNA Technologies, Inc., Coralville, IA, USA.
  • ,
  • Rolf Turk, Integrated DNA Technologies, Inc., Coralville, IA, USA.
  • ,
  • Ashley M Jacobi, Integrated DNA Technologies, Inc., Coralville, IA, USA.
  • ,
  • Michael A Collingwood, Integrated DNA Technologies, Inc., Coralville, IA, USA.
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  • Nicole M Bode, Integrated DNA Technologies, Inc., Coralville, IA, USA.
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  • Matthew S McNeill, Integrated DNA Technologies, Inc., Coralville, IA, USA.
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  • Shuqi Yan, Integrated DNA Technologies, Inc., Coralville, IA, USA.
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  • Joab Camarena, Department of Pediatrics, Stanford University, Stanford, California, USA.
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  • Ciaran M Lee, Department of Bioengineering, Rice University, Houston, TX, USA.
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  • So Hyun Park, Department of Bioengineering, Rice University, Houston, TX, USA.
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  • Volker Wiebking, Department of Pediatrics, Stanford University, Stanford, California, USA.
  • ,
  • Rasmus O Bak
  • Natalia Gomez-Ospina, Department of Pediatrics, Stanford University, Stanford, California, USA.
  • ,
  • Mara Pavel-Dinu, Department of Pediatrics, Stanford University, Stanford, California, USA.
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  • Wenchao Sun, Biomaterials and Advanced Drug Delivery Laboratory, Stanford University School of Medicine, Palo Alto, CA, USA.
  • ,
  • Gang Bao, Department of Bioengineering, Rice University, Houston, TX, USA.
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  • Matthew H Porteus, Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.
  • ,
  • Mark A Behlke, Integrated DNA Technologies, Inc., Coralville, IA, USA. mbehlke@idtdna.com.

Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

Original languageEnglish
JournalNature Medicine
Volume24
Issue8
Pages (from-to)1216-1224
Number of pages9
ISSN1078-8956
DOIs
Publication statusPublished - 2018

    Research areas

  • BONE-MARROW, CRISPR-CAS9 NUCLEASES, GENOME, GUIDE RNA, HUMAN T-CELLS, IN-VIVO, MAMMALIAN-CELLS, MARROW TRANSPLANTATION, MESSENGER-RNA, OFF-TARGET

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