title: Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.
abstract: Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA.
authors.forename: Pankaj K
authors.initials: PK
authors.lastname: Mandal
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Leonardo M R
authors.initials: LM
authors.lastname: Ferreira
authors.affiliation: Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Collins
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Torsten B
authors.initials: TB
authors.lastname: Meissner
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA.
authors.forename: Christian L
authors.initials: CL
authors.lastname: Boutwell
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Max
authors.initials: M
authors.lastname: Friesen
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Vrbanac
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Brian S
authors.initials: BS
authors.lastname: Garrison
authors.affiliation: Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Alexei
authors.initials: A
authors.lastname: Stortchevoi
authors.affiliation: Institution for Experimental Medical Research, Immunology section, Lund University, 221 84, Lund, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: Bryder
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Musunuru
authors.affiliation: Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Harrison
authors.initials: H
authors.lastname: Brand
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Tager
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA.
authors.forename: Todd M
authors.initials: TM
authors.lastname: Allen
authors.affiliation: Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA; Broad Institute, Cambridge, MA 02142, USA; Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Talkowski
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address: derrick.rossi@childrens.harvard.edu.
authors.forename: Derrick J
authors.initials: DJ
authors.lastname: Rossi
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute, Cambridge, MA 02142, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address: chad_cowan@harvard.edu.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Cowan
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D019070:Cell Lineage; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D017353:Gene Deletion; D018390:Gene Targeting; D056426:Genetic Loci; D015894:Genome, Human / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D017393:RNA Editing / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; C584933:CCR5 protein, human; D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D019713:Receptors, CCR5