title: CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo-Brief Report.
abstract: Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology-permanent alteration of genomic DNA sequences-mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients.
  
  
  
  We used a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis.
  
  
  
  This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.).
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.).
authors.forename: Avanthi
authors.initials: A
authors.lastname: Raghavan
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.).
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.).
authors.forename: Lyon
authors.initials: L
authors.lastname: Qiao
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.).
authors.forename: Yongxian
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.). kiranmusunuru@gmail.com qrding@sibs.ac.cn.
authors.forename: Qiurong
authors.initials: Q
authors.lastname: Ding
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (X.W., A.R., T.C., L.Q., K.M.); Harvard Medical School, Boston, MA (A.R., K.M.); Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China (Y.Z., Q.D.); Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (K.M.); and Broad Institute, Cambridge, MA (K.M.). kiranmusunuru@gmail.com qrding@sibs.ac.cn.
authors.forename: Kiran
authors.initials: K
authors.lastname: Musunuru
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics; D015536:Down-Regulation; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D005838:Genotype; D022781:Hepatocytes / Q000201:enzymology* / Q000637:transplantation; D006801:Humans; D006867:Hydrolases / Q000172:deficiency / Q000235:genetics; D053631:Interleukin Receptor Common gamma Subunit / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D000071449:Proprotein Convertase 9 / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D004268:DNA-Binding Proteins; C507942:Il2rg protein, mouse; D053631:Interleukin Receptor Common gamma Subunit; C499308:Rag2 protein, mouse; D006867:Hydrolases; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; C020156:fumarylacetoacetase
keywords: gene therapy; liver; molecular biology; mutagenesis; subtilisins