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Nature Biotechnology Jan 2024Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an...
Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.
Topics: Humans; CRISPR-Cas Systems; Transposases; Plasmids; DNA; Genome; Gene Editing
PubMed: 36991112
DOI: 10.1038/s41587-023-01748-1 -
Circulation Apr 2024Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of...
BACKGROUND
Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined.
METHODS
In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction.
RESULTS
The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of , its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function.
CONCLUSIONS
In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.
Topics: Humans; Animals; Mice; Gene Regulatory Networks; Chromatin Assembly and Disassembly; Chromatin; Myocytes, Cardiac; Heart Failure; RNA; Transposases; ATPases Associated with Diverse Cellular Activities; DNA-Binding Proteins
PubMed: 38152931
DOI: 10.1161/CIRCULATIONAHA.123.065440 -
Clinical Infectious Diseases : An... Sep 2023Integrase strand transfer inhibitors (INSTIs) have been associated with an increased risk for cardiovascular disease (CVD) events. We investigated the impact of starting... (Clinical Trial)
Clinical Trial
BACKGROUND
Integrase strand transfer inhibitors (INSTIs) have been associated with an increased risk for cardiovascular disease (CVD) events. We investigated the impact of starting INSTI-based antiretroviral therapy (ART) on CVD events among treatment-naïve people with human immunodeficiency virus using a target trial framework, which reduces the potential for confounding and selection bias.
METHODS
We included Swiss HIV Cohort Study participants who were ART-naïve after May 2008, when INSTIs became available in Switzerland. Individuals were categorized according to their first ART regimen (INSTI vs other ART) and were followed from ART start until the first of CVD event (myocardial infarction, stroke, or invasive cardiovascular procedure), loss to follow-up, death, or last cohort visit. We calculated hazard ratios and risk differences using pooled logistic regression models with inverse probability of treatment and censoring weights.
RESULTS
Of 5362 participants (median age 38 years, 21% women, 15% of African origin), 1837 (34.3%) started INSTI-based ART, and 3525 (65.7%) started other ART. Within 4.9 years (interquartile range, 2.4-7.4), 116 CVD events occurred. Starting INSTI-based ART was not associated with an increased risk for CVD events (adjusted hazard ratio, 0.80; 95% confidence interval [CI], .46-1.39). Adjusted risk differences between individuals who started INSTIs and those who started other ART were -0.17% (95% CI, -.37 to .19) after 1 year, -0.61% (-1.54 to 0.22) after 5 years, and -0.71% (-2.16 to 0.94) after 8 years.
CONCLUSIONS
In this target trial emulation, we found no difference in short- or long-term risk for CVD events between treatment-naïve people with human immunodeficiency virus who started INSTI-based ART and those on other ART.
Topics: Adult; Female; Humans; Male; Anti-HIV Agents; Cardiovascular Diseases; Cohort Studies; HIV; HIV Infections; HIV Integrase Inhibitors
PubMed: 37157869
DOI: 10.1093/cid/ciad286 -
Molecular Therapy. Nucleic Acids Mar 2024Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion... (Review)
Review
Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.
PubMed: 38379727
DOI: 10.1016/j.omtn.2024.102138 -
Cell Reports Sep 2023Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has...
Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.
Topics: Animals; Mice; Microglia; Integrases; Gene Transfer Techniques; Mice, Transgenic
PubMed: 37635351
DOI: 10.1016/j.celrep.2023.113031 -
Frontiers in Chemistry 2024Biological membranes consist of integral and peripheral protein-associated lipid bilayers. Although constituent lipids vary among cells, membrane lipids are mainly... (Review)
Review
Biological membranes consist of integral and peripheral protein-associated lipid bilayers. Although constituent lipids vary among cells, membrane lipids are mainly classified as phospholipids, glycolipids, and sterols. Phospholipids are further divided into glycerophospholipids and sphingophospholipids, whereas glycolipids are further classified as glyceroglycolipids and sphingoglycolipids. Both glycerophospholipids and glyceroglycolipids contain diacylglycerol as the common backbone, but their head groups differ. Most glycerolipids have polar head groups containing phosphate esters or sugar moieties. However, trace components termed glycero-glycophospholipids, each possessing both a phosphate ester and a sugar moiety, exist in membranes. Recently, the unique biological activities of glycero-glycophospholipids have attracted considerable attention. In this review, we describe the structure, distribution, function, biosynthesis, and chemical synthetic approaches of representative glycero-glycophospholipids-phosphatidylglucoside (PtdGlc) and enterobacterial common antigen (ECA). In addition, we introduce our recent studies on the rare glycero-glyco"pyrophospho"lipid, membrane protein integrase (MPIase), which is involved in protein translocation across biomembranes.
PubMed: 38389730
DOI: 10.3389/fchem.2024.1353688 -
International Journal of Molecular... Dec 2023Prime editing is a rapidly developing method of CRISPR/Cas-based genome editing. The increasing number of novel PE applications and improved versions demands constant... (Review)
Review
Prime editing is a rapidly developing method of CRISPR/Cas-based genome editing. The increasing number of novel PE applications and improved versions demands constant analysis and evaluation. The present review covers the mechanism of prime editing, the optimization of the method and the possible next step in the evolution of CRISPR/Cas9-associated genome editing. The basic components of a prime editing system are a prime editor fusion protein, consisting of nickase and reverse transcriptase, and prime editing guide RNA, consisting of a protospacer, scaffold, primer binding site and reverse transcription template. Some prime editing systems include other parts, such as additional RNA molecules. All of these components were optimized to achieve better efficiency for different target organisms and/or compactization for viral delivery. Insights into prime editing mechanisms allowed us to increase the efficiency by recruiting mismatch repair inhibitors. However, the next step in prime editing evolution requires the incorporation of new mechanisms. Prime editors combined with integrases allow us to combine the precision of prime editing with the target insertion of large, several-kilobase-long DNA fragments.
Topics: RNA, Guide, CRISPR-Cas Systems; Binding Sites; DNA Mismatch Repair; Deoxyribonuclease I; Gene Editing; CRISPR-Cas Systems
PubMed: 38069367
DOI: 10.3390/ijms242317045 -
PloS One 2023Integrase strand transfer inhibitors (INSTI) are one of the most prescribed drug classes for the treatment of HIV infection worldwide. Emtricitabine/Tenofovir...
INTRODUCTION
Integrase strand transfer inhibitors (INSTI) are one of the most prescribed drug classes for the treatment of HIV infection worldwide. Emtricitabine/Tenofovir Alafenamide/ Bictegravir (FTC/TAF/BIC) has been evaluated in randomized clinical trials; few studies have verified tolerability and safety in clinical practice. Our aim was to investigate the metabolic and hepatic safety in a real-life setting of FTC/TAF/BIC.
MATERIALS AND METHODS
Consecutive people living with HIV infection (PLWH) enrolled in the SCOLTA project, switching to or initiating their first antiretroviral treatment with FTC/TAF/BIC were included. PLWH with HBV co-infection were excluded. Metabolic and hepatic variables were collected at T0 and T1, were defined as baseline and 6-month follow-up respectively, and their modifications were analysed using the paired t-test and the analysis of variance.
RESULTS
Five hundred and thirty-nine PLWH with at least one follow-up visit were included in the analysis. Mean age was 48 years (±12.1), 74% were male, 16.1% were naïve to antiretrovirals (ART). At T1, ART-experienced PLWH showed a significant reduction of total cholesterol (TC) and triglycerides, and a slight increase in blood glucose (BG) and ALT. On the contrary, in ART-naïve PLWH blood lipids significantly increased, although with an unaffected TC/high density lipoprotein (HDL)-c ratio, while alanine aminotransferase (ALT) decreased significantly, mainly in those with altered baseline level. The treatment interruptions were 45 (8.4%) over the whole observation period, 13 (2.4%) due to AEs. The most frequent AEs were related to the central nervous system (6 events of depression, insomnia, headache, agitation) and 3 PLWH discontinued the regimen because of grade 1-2 weight gain.
CONCLUSIONS
In ART-experienced PLWH switching to FTC/TAF/BIC a significant improvement of lipid profile occurred but with significant BG and ALT variation without clinical relevance. In ART-naïve PLWH, blood lipids increased even though lipid profile did not worsen, and a trend towards normalization of liver enzymes was suggested. FTC/TAF/BIC is well tolerated in the real life setting.
Topics: Male; Humans; Middle Aged; Female; HIV Infections; Anti-HIV Agents; Emtricitabine; Alanine; Heterocyclic Compounds, 3-Ring; Pyridones; Anti-Retroviral Agents; Lipoproteins, HDL
PubMed: 37556481
DOI: 10.1371/journal.pone.0289132 -
Science Advances Jul 2023HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand...
HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.
Topics: HIV Integrase Inhibitors; Oxazines; Mutation; HIV Integrase
PubMed: 37478179
DOI: 10.1126/sciadv.adg5953