A programmable method for transcriptional tuning in mammalian cells is presented and an orthogonal platform to dissect the mechanistic function of chromatin regulators in living cells is offered.
Abstract
The ability to control the expression of human genes is a major goal in synthetic biology, enables dissection of gene function, and can be harnessed for therapeutic applications. Advances in genome editing and transcriptional engineering often result in complete gene inactivation or full transcriptional repression. However, programmable tools to dial transcription at intermediate levels remain challenging. Here, we present CRISPRtune – a synthetic fusion of MeCP2 to catalytically dead dCas9 that tunes down transcription of endogenous genes in human cells by harnessing the mild repressor activity of MeCP2. Using pooled genome-scale CRISPR screens, we tune the expression of thousands of endogenous genes and define the targeting rules of CRISPRtune in human cells. With a platform to target MeCP2 at defined genomic sites, we show the direct epigenetic changes induced by MeCP2 at gene promoters and we identify its genetic dependency partners for productive transcriptional repression. Rett syndrome-associated mutations of MeCP2 show defects for transcriptional repression due to their failure to remodel the local epigenetic landscape of target genes. Together, we present a programmable method for transcriptional tuning in mammalian cells and offer an orthogonal platform to dissect the mechanistic function of chromatin regulators in living cells.
The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation and makes biomolecular condensation a general principle for enhancing CRISPR gene regulation.
Aolin Li, Congcong Cao, Chunyan Yang et al.· Theranostics· 0 citations
This chapter outlines a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.
Ananya Mukherjee, Shrabani Basak, Raghuvir Singh et al.· Methods in molecular biology· 0 citations
This review comprehensively discusses the molecular mechanisms underlying epigenetic regulation, key epigenome editing tools, their modes of action, and the emerging applications of epigenome editing in plant biology, highlighting it as a potential tool for sustainable agriculture and precision breeding.
Marifa Gulzar, Gazala Nazir, F. Mushtaq et al.· Saudi Journal of Food Securi...· 0 citations
The gain or loss of chromosomes in eukaryotes often drives aberrant phenotypes by altering the expression levels of hundreds or thousands of genes. In the case of beneficial aneuploidies, the genetic basis of fitness improvement has rarely been pinpointed, and identifying the causal genes remains a major challenge in engineering biology. The leading cause of human fungal infections, Candida albicans, frequently acquires extra copies of chromosome R (ChrR) following exposure to azole antifungal drugs, resulting in heightened antifungal tolerance. Here, we combine RNA-seq with parallel chromosome-wide CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens to systematically profile the ChrR genes contributing to azole tolerance. Using multiplexed CRISPR-dCas12a tools, we further characterize the combinatorial effects of candidate genes and uncover a central role for post-Golgi secretory trafficking in antifungal tolerance. Specifically, we demonstrate that the secretory pathway regulators SEC4 and YPT31 are both necessary and sufficient for ChrR-mediated azole tolerance. By leveraging a large-scale CRISPRa screen in a fungal pathogen, our work functionally dissects one of the most common aneuploidies observed in C. albicans, provides mechanistic insight into the molecular basis of antifungal tolerance, and establishes a generalizable framework for studying aneuploidy-mediated phenotypes across eukaryotic organisms.
Nicholas C. Gervais, Lauren F. Wensing, Manini Chhina et al.· bioRxiv· 0 citations
Two inducible CRISPR-based perturbation platforms are characterized in K562 human hematopoietic cells: dual-inducible CRISPRi system employing a dCas9–KRAB–DHFR–TetON architecture controlled by doxycycline and trimethoprim and single-inducible CRISPRa system employing a dCas9–VPR–TetON architecture controlled by Dox alone.
To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· Nature Communications· 0 citations