Massively parallel base editing to map variant effects in human hematopoiesis

Jorge D Martin-Rufino; Nicole Castano; Michael Pang; Emanuelle I Grody; Samantha Joubran; Alexis Caulier; Lara Wahlster; Tongqing Li; Xiaojie Qiu; Anna Maria Riera-Escandell; Gregory A Newby; Aziz Al'Khafaji; Santosh Chaudhary; Susan Black; Chen Weng; Glen Munson; David R Liu; Marcin W Wlodarski; Kacie Sims; Jamie H Oakley; Ross M Fasano; Ramnik J Xavier; Eric S Lander; Daryl E Klein; Vijay G Sankaran

doi:10.1016/j.cell.2023.03.035

Massively parallel base editing to map variant effects in human hematopoiesis

Cell. 2023 May 25;186(11):2456-2474.e24. doi: 10.1016/j.cell.2023.03.035. Epub 2023 May 2.

Authors

Jorge D Martin-Rufino¹, Nicole Castano², Michael Pang³, Emanuelle I Grody⁴, Samantha Joubran⁵, Alexis Caulier², Lara Wahlster², Tongqing Li⁶, Xiaojie Qiu⁷, Anna Maria Riera-Escandell⁸, Gregory A Newby⁹, Aziz Al'Khafaji⁴, Santosh Chaudhary⁴, Susan Black², Chen Weng¹⁰, Glen Munson⁴, David R Liu⁹, Marcin W Wlodarski¹¹, Kacie Sims¹², Jamie H Oakley¹³, Ross M Fasano¹³, Ramnik J Xavier¹⁴, Eric S Lander⁴, Daryl E Klein⁶, Vijay G Sankaran¹⁵

Affiliations

¹ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; PhD Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA.
² Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard-MIT Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁵ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Chemical Biology PhD Program, Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Pharmacology and Yale Cancer Biology Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
⁷ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
⁸ Independent Researcher, now at Google, Zurich, Switzerland.
⁹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
¹⁰ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
¹¹ Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
¹² St. Jude Affiliate Clinic at Our Lady of the Lake Children's Health, Baton Rouge, LA 70809, USA.
¹³ Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA.
¹⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Computational and Integrative Biology, Department of Molecular Biology, and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
¹⁵ Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address: [email protected].

Abstract

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

Keywords: base editing; differentiation; functional screens; genome engineering; hematopoiesis; hematopoietic stem cell; primary cells; single-cell genomics.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems
Cell Differentiation
Gene Editing*
Genetic Engineering
Genome
Hematopoiesis
Hematopoietic Stem Cells* / metabolism
Humans
Single-Cell Analysis

Abstract

Publication types

MeSH terms

Grants and funding