title: Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.
abstract: Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.
authors.affiliation: Department of Medicine and Institute for Human Genetics, lin.ye@ucsf.edu yw.kan@ucsf.edu.
authors.forename: Lin
authors.initials: L
authors.lastname: Ye
authors.affiliation: Department of Medicine and Institute for Human Genetics,Department of Laboratory Medicine, and.
authors.forename: Jiaming
authors.initials: J
authors.lastname: Wang
authors.affiliation: Blood Systems Research Institute, San Francisco, CA 94118; and.
authors.forename: Ashley I
authors.initials: AI
authors.lastname: Beyer
authors.affiliation: Department of Medicine and Institute for Human Genetics,Division of Hematology/Oncology, University of California, San Francisco, CA 94143;
authors.forename: Fernando
authors.initials: F
authors.lastname: Teque
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cradick
authors.affiliation: Department of Laboratory Medicine, and.
authors.forename: Zhongxia
authors.initials: Z
authors.lastname: Qi
authors.affiliation: Department of Medicine and Institute for Human Genetics.
authors.forename: Judy C
authors.initials: JC
authors.lastname: Chang
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Department of Laboratory Medicine, andBlood Systems Research Institute, San Francisco, CA 94118; andThe Liver Center.
authors.forename: Marcus O
authors.initials: MO
authors.lastname: Muench
authors.affiliation: Department of Laboratory Medicine, and.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Yu
authors.affiliation: Division of Hematology/Oncology, University of California, San Francisco, CA 94143;
authors.forename: Jay A
authors.initials: JA
authors.lastname: Levy
authors.affiliation: Department of Medicine and Institute for Human Genetics,Department of Laboratory Medicine, and lin.ye@ucsf.edu yw.kan@ucsf.edu.
authors.forename: Yuet Wai
authors.initials: YW
authors.lastname: Kan
mesh_terms: D015139:Blotting, Southern; D002454:Cell Differentiation / Q000276:immunology*; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism; D060467:Disease Resistance / Q000235:genetics* / Q000276:immunology; D005455:Fluorescent Antibody Technique; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D015658:HIV Infections / Q000235:genetics* / Q000276:immunology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000276:immunology*; D008264:Macrophages / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D016296:Mutagenesis / Q000235:genetics; D019713:Receptors, CCR5 / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics*; D019895:Transposases / Q000378:metabolism
chemical_list: C584933:CCR5 protein, human; D017931:DNA Primers; D019713:Receptors, CCR5; D019895:Transposases; D003851:Deoxyribonucleases
keywords: TTAA site; cellular therapy; homologous recombination; off-site target