RESUMO
CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Genoma Humano , Células-Tronco Hematopoéticas/metabolismo , RNA Guia de Cinetoplastídeos/genética , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Células HEK293 , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Interações entre Hospedeiro e Microrganismos/genética , Proteínas Nucleares/genética , Antígenos de Superfície/genética , Antivirais/farmacologia , Linhagem Celular Tumoral , DNA Viral/genética , Estudo de Associação Genômica Ampla/métodos , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Humanos , Polinucleotídeo Adenililtransferase/genética , Carga Viral/efeitos dos fármacos , Carga Viral/genéticaRESUMO
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.