RESUMO
Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.
Assuntos
Edição de Genes , Receptores CCR5 , Receptores CXCR4 , Edição de Genes/métodos , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/terapia , HIV-1/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Linfócitos T/metabolismoRESUMO
Designer effectors based on the DNA binding domain (DBD) of Xanthomonas transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive structure of the TALE arrays composing the DBD impedes their generation as gene synthesis product and prevents the delivery of TALE-based genes using lentiviral vectors (LVs), a widely used system for human gene therapy. To overcome these limitations, we aimed at chimerizing the DNA sequence encoding for the TALE-DBDs by introducing sufficient diversity to facilitate both their gene synthesis and enable their lentiviral delivery. To this end, we replaced three out of 17 Xanthomonas TALE repeats with TALE-like units from the bacterium Burkholderia rhizoxinica. This was combined with extensive codon variation and specific amino acid substitutions throughout the DBD in order to maximize intra- and inter-repeat sequence variability. We demonstrate that chimerized TALEs can be easily generated using conventional Golden Gate cloning strategy or gene synthesis. Moreover, chimerization enabled the delivery of TALE-based designer nucleases, transcriptome and epigenome editors using lentiviral vectors. When delivered as plasmid DNA, chimerized TALEs targeting the CCR5 and CXCR4 loci showed comparable activities in human cells. However, lentiviral delivery of TALE-based transcriptional activators was only successful in the chimerized form. Similarly, delivery of a chimerized CXCR4-specific epigenome editor resulted in rapid silencing of endogenous CXCR4 expression. In conclusion, extensive codon variation and chimerization of TALE-based DBDs enables both the simplified generation and the lentiviral delivery of designer TALEs, and therefore facilitates the clinical application of these tools to precisely edit the genome, transcriptome and epigenome.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Lentivirus/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Efetores Semelhantes a Ativadores de Transcrição/química , Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.
Assuntos
Epigenoma/genética , Epigenômica/métodos , Edição de Genes/métodos , Sistemas CRISPR-Cas , Linhagem Celular , DNA/genética , Metilação de DNA/genética , Epigênese Genética/genética , Genes Reporter/genética , Genoma/genética , Células HEK293 , HumanosRESUMO
Liver fibrosis is a major disease that is primarily caused by hepatitis virus infections, toxins, and alcohol abuse. Diagnosing and staging liver fibrosis are critical in guiding the treatment of chronic liver diseases, according to several international and Chinese guidelines. Liver biopsy is the gold standard for diagnosing and staging liver fibrosis, but it is invasive and suffers from several limitations. Consequently, much research has focused on the search for a noninvasive serum biomarker of fibrosis. In this study, we determined that Chitinase 3-like 1 (CHI3L1) is an abundantly expressed liver gene whose expression is highly enriched in the liver. We then compared serum levels of CHI3L1 among patients with various stages of liver fibrosis, as determined by liver biopsies, and found that the CHI3L1 levels were able to differentiate early stages of liver fibrosis (S0-S2) from late stages of liver fibrosis (S3-S4). We further showed that CHI3L1 is a good marker of substantial fibrosis, with areas under the ROC curves (AUCs) of 0.94 for substantial (S2, S3, S4) fibrosis and 0.96 for advanced (S3, S4) fibrosis. Finally, we showed that CHI3L1 is superior to hyaluronic acid (HA), type III procollagen (PCIII), laminin (LN), and type IV collagen (CIV), which are also serum biomarkers of liver fibrosis, in identifying advanced liver fibrosis in patients with HBV-related liver fibrosis in China.