A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells.

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

[1]  T. Masuda,et al.  Identification of a Novel Human Immunodeficiency Virus Type 1 Integrase Interactor, Gemin2, That Facilitates Efficient Viral cDNA Synthesis In Vivo , 2006, Journal of Virology.

[2]  Paul E. Kennedy,et al.  HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor , 1996, Science.

[3]  Kenneth A. Matreyek,et al.  The Requirement for Nucleoporin NUP153 during Human Immunodeficiency Virus Type 1 Infection Is Determined by the Viral Capsid , 2011, Journal of Virology.

[4]  Wulin Teo,et al.  An Essential Role for LEDGF/p75 in HIV Integration , 2006, Science.

[5]  E. Thiel,et al.  Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. , 2009, The New England journal of medicine.

[6]  A. Graessmann,et al.  A synthetic peptide bearing the HIV-1 integrase 161-173 amino acid residues mediates active nuclear import and binding to importin alpha: characterization of a functional nuclear localization signal. , 2004, Journal of molecular biology.

[7]  Richard A Koup,et al.  Homozygous Defect in HIV-1 Coreceptor Accounts for Resistance of Some Multiply-Exposed Individuals to HIV-1 Infection , 1996, Cell.

[8]  M. Llano,et al.  Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75. , 2006, Journal of molecular biology.

[9]  Anitha Rao,et al.  RNA-Based Gene Therapy for HIV with Lentiviral Vector–Modified CD34+ Cells in Patients Undergoing Transplantation for AIDS-Related Lymphoma , 2010, Science Translational Medicine.

[10]  David A. Scott,et al.  Rationally engineered Cas9 nucleases with improved specificity , 2015, Science.

[11]  S. Ekker,et al.  TALEN Knockout of the PSIP1 Gene in Human Cells: Analyses of HIV-1 Replication and Allosteric Integrase Inhibitor Mechanism , 2014, Journal of Virology.

[12]  R. Doms,et al.  Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. , 2014, Blood.

[13]  D. Price.  P-TEFb, a Cyclin-Dependent Kinase Controlling Elongation by RNA Polymerase II , 2000, Molecular and Cellular Biology.

[14]  Amy S. Espeseth,et al.  Genome-scale RNAi screen for host factors required for HIV replication. , 2008, Cell host & microbe.

[15]  Sourav Bandyopadhyay,et al.  Quantitative genetic-interaction mapping in mammalian cells , 2013, Nature Methods.

[16]  K. Ullman,et al.  Versatility at the nuclear pore complex: lessons learned from the nucleoporin Nup153 , 2005, Chromosoma.

[17]  Fulvio Mavilio,et al.  Gene therapy , 1993, Nature.

[18]  C. Van den Haute,et al.  Transient and Stable Knockdown of the Integrase Cofactor LEDGF/p75 Reveals Its Role in the Replication Cycle of Human Immunodeficiency Virus , 2006, Journal of Virology.

[19]  Zeger Debyser,et al.  Transportin-SR2 Imports HIV into the Nucleus , 2008, Current Biology.

[20]  John H. Morris,et al.  Global landscape of HIV–human protein complexes , 2011, Nature.

[21]  Wei-Ting Hwang,et al.  Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. , 2014, The New England journal of medicine.

[22]  S. Antonarakis,et al.  TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm , 2013, Retrovirology.

[23]  John P. Moore,et al.  The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. , 2004, AIDS research and human retroviruses.

[24]  Infection Through a Functional Genomic Screen Identification of Host Proteins Required for HIV , 2008 .

[25]  Marc Parmentier,et al.  Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene , 1996, Nature.

[26]  J. Orange,et al.  Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases , 2008, Nature Biotechnology.

[27]  D. Baltimore.  Gene therapy. Intracellular immunization. , 1988, Nature.

[28]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[29]  D. Hazuda,et al.  P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. , 1997, Genes & development.

[30]  B. Torbett,et al.  Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[31]  J. Rain,et al.  The integrase cofactor LEDGF/p75 associates with Iws1 and Spt6 for postintegration silencing of HIV-1 gene expression in latently infected cells. , 2015, Cell host & microbe.

[32]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[33]  Guangfeng Song,et al.  HIV-1, human interaction database: current status and new features , 2014, Nucleic Acids Res..

[34]  J. Lieberman,et al.  Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen , 2007, Science.

[35]  G. Kalpana,et al.  Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance* , 2009, The Journal of Biological Chemistry.

[36]  C. Broder,et al.  CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. , 1996, Science.

[37]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[38]  A. Fire,et al.  Landscape of target:guide homology effects on Cas9-mediated cleavage , 2014, Nucleic acids research.

[39]  R Brookmeyer,et al.  Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. , 1997, Science.

[40]  Joseph Sodroski,et al.  CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5 , 1996, Nature.

[41]  D. McDermott,et al.  CCR5 deficiency increases risk of symptomatic West Nile virus infection , 2006, The Journal of experimental medicine.

[42]  S. Goff,et al.  Host factors exploited by retroviruses , 2007, Nature Reviews Microbiology.

[43]  Stephen C. Peiper,et al.  Identification of a major co-receptor for primary isolates of HIV-1 , 1996, Nature.

[44]  Jennifer A. Doudna,et al.  Generation of knock-in primary human T cells using Cas9 ribonucleoproteins , 2015, Proceedings of the National Academy of Sciences.

[45]  J. Rain,et al.  Inhibition of Early Steps of HIV-1 Replication by SNF5/Ini1* , 2006, Journal of Biological Chemistry.

[46]  Paul Shinn,et al.  A role for LEDGF/p75 in targeting HIV DNA integration , 2005, Nature Medicine.

[47]  Mark A. Kay,et al.  Progress and problems with the use of viral vectors for gene therapy , 2003, Nature Reviews Genetics.

[48]  M. Llano,et al.  LEDGF/p75 Determines Cellular Trafficking of Diverse Lentiviral but Not Murine Oncoretroviral Integrase Proteins and Is a Component of Functional Lentiviral Preintegration Complexes , 2004, Journal of Virology.

[49]  Masahiko Kuroda,et al.  Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development , 1998, Nature.

[50]  J. McCune,et al.  Can HIV be cured with stem cell therapy? , 2010, Nature Biotechnology.

[51]  C. Körber,et al.  Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes , 2007, Journal of neuroscience research.

[52]  Sumit K Chanda,et al.  Identifying HIV-1 host cell factors by genome-scale RNAi screening. , 2011, Methods.

[53]  T. Albert,et al.  Characterization of molecular and cellular functions of the cyclin‐dependent kinase CDK9 using a novel specific inhibitor , 2014, British journal of pharmacology.

[54]  M. Mathews,et al.  Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. , 1997, Genes & development.

[55]  R. Connor,et al.  Change in Coreceptor Use Correlates with Disease Progression in HIV-1–Infected Individuals , 1997, The Journal of experimental medicine.

[56]  D. Baltimore.  Intracellular immunization , 1988, Nature.

[57]  Jeffrey C. Miller,et al.  An unbiased genome-wide analysis of zinc-finger nuclease specificity , 2011, Nature Biotechnology.

[58]  A. Friedler,et al.  Transportin 3 and importin α are required for effective nuclear import of HIV-1 integrase in virus-infected cells , 2010, Nucleus.

[59]  Vanessa Taupin,et al.  Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo , 2010, Nature Biotechnology.

[60]  J. Church.  Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen , 2008, Pediatrics.

[61]  J. Keith Joung,et al.  731. High-Fidelity CRISPR-Cas9 Nucleases with No Detectable Genome-Wide Off-Target Effects , 2016 .

[62]  J. Riley,et al.  Engineering T Cells to Functionally Cure HIV-1 Infection. , 2015, Molecular therapy : the journal of the American Society of Gene Therapy.

[63]  E. Thiel,et al.  Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. , 2011, Blood.

[64]  R. König,et al.  Global Analysis of Host-Pathogen Interactions that Regulate Early-Stage HIV-1 Replication , 2008, Cell.

[65]  M. Porteus,et al.  Generation of an HIV resistant T-cell line by targeted "stacking" of restriction factors. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[66]  Lluis Montoliu,et al.  Genome Editing , 2018, Advances in Experimental Medicine and Biology.

[67]  C. Broder,et al.  CC CKR5: A RANTES, MIP-1α, MIP-1ॆ Receptor as a Fusion Cofactor for Macrophage-Tropic HIV-1 , 1996, Science.

[68]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[69]  Masashi Miyano,et al.  Augmentation of Reverse Transcription by Integrase through an Interaction with Host Factor, SIP1/Gemin2 Is Critical for HIV-1 Infection , 2009, PloS one.

[70]  M. Jinek,et al.  In vitro enzymology of Cas9. , 2014, Methods in enzymology.

[71]  Anders Karlsson,et al.  Maraviroc for previously treated patients with R5 HIV-1 infection. , 2008, The New England journal of medicine.

[72]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[73]  D. Weissman,et al.  Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[74]  B. de Strooper,et al.  DNA-Dependent Protein Kinase Is Not Required for Efficient Lentivirus Integration , 2000, Journal of Virology.

[75]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[76]  A. Engelman,et al.  LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. , 2007, Genes & development.

[77]  A. Jordan,et al.  The site of HIV‐1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation , 2001, The EMBO journal.

[78]  David R. Liu,et al.  Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection , 2011, Nature Methods.

[79]  B. Fahrenkrog,et al.  Structural characterization of altered nucleoporin Nup153 expression in human cells by thin-section electron microscopy , 2014, Nucleus.

[80]  A. Engelman,et al.  Biochemical and genetic analyses of integrase-interacting proteins lens epithelium-derived growth factor (LEDGF)/p75 and hepatoma-derived growth factor related protein 2 (HRP2) in preintegration complex function and HIV-1 replication. , 2006, Virology.

[81]  R. Siliciano,et al.  Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells , 2003, Nature Medicine.

[82]  J. Bourhis,et al.  Effect of Ku80 depletion on the preintegrative steps of HIV-1 replication in human cells. , 2002, Virology.

[83]  F. Bushman,et al.  Role of the non‐homologous DNA end joining pathway in the early steps of retroviral infection , 2001, The EMBO journal.

[84]  David Bryder,et al.  Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. , 2014, Cell stem cell.

[85]  Gabriela Plesa,et al.  Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. , 2013, Human gene therapy.

[86]  F. Bushman,et al.  Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases , 2011, PLoS pathogens.

[87]  T. Hope,et al.  HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[88]  M. Giacca,et al.  GCN5-dependent acetylation of HIV-1 integrase enhances viral integration , 2009, Retrovirology.

[89]  I. Vitale,et al.  Impact of the Ku Complex on HIV-1 Expression and Latency , 2013, PloS one.

[90]  Jang-Gi Choi,et al.  CCR5 Gene Editing of Resting CD4+ T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice , 2014, Molecular therapy. Nucleic acids.

[91]  D. Richman,et al.  Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. , 1997, Science.

[92]  R. Doms,et al.  A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains , 1996, Journal of virology.