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Understanding how gene expression impacts plant development and physiology is important for crop engineering. Programmable transcriptional activators (PTAs), including CRISPR-Cas activators, have relied on a limited number of transcriptional activation domains (ADs) (Casas-Mollano et al., 2020; Lowder et al., 2018; Pan et al., 2021; Papikian et al., 2019). Usually, the VP64 domain, derived from herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. In dCas9-based PTAs, binding to a target DNA sequence is afforded by the dCas9-sgRNA ribonucleoprotein. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived AD.

Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) technology allows pooled or targeted upregulation of gene expression. This new technical capacity helps to identify genes that, when upregulated, modify cell physiology and/or disease progression.Various CRISPR activation strategies have been described, with single and combinatorial activator domains enhancing gene activation efficiency. Different activation domains are suited to specific genes and cell types.CRISPRa pooled screening has enabled diverse applications in drug discovery, providing new insight into cell responses and cell differentiation, and even an understanding of SARS-CoV-2 mechanisms during infection.CRISPRa also demonstrates strong potential for in vivo applications for various diseases and conditions. Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening. Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery to achieve greater, more robust, and more consistent activation is continually under development. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

High-throughput genetic screens interfering with gene expression are invaluable tools to identify gene function and phenotype-to-genotype interactions. Implementing such screens in the laboratory is challenging, and the choice between currently available technologies based on RNAi and CRISPR/Cas9 (CRISPR-associated protein 9) is not trivial. Identifying reliable candidate hits requires a streamlined experimental setup adjusted to the specific biological question. Here, we provide a critical assessment of the various RNAi/CRISPR approaches to pooled screens and discuss their advantages and pitfalls. We specify a set of best practices for key parameters enabling a reproducible screen and provide a detailed overview of analysis methods and repositories for identifying the best candidate gene hits. Pooled genetic screens based on RNAi and CRISPR technologies are a powerful approach for high-throughput interrogation of loss- or gain-of-function and phenotype-to-genotype correlations. Several CRISPR technologies are applicable for pooled screens, allowing for a wide range of genetic perturbations and mutagenesis beyond classical RNAi-based gene knockdown. Stringent experimental design, appropriate controls and careful library selection are essential to identify valid hits. Appropriate library representation throughout the screening procedure is key to avoid false positives/negatives. Different bioinformatics pipelines can be applied to data analysis, and their combination may lead to increased specificity of selected hits.

Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock‐in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV‐based strategy carrying intein‐split‐dCas9 in combination with activators (AAV‐dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single‐cell transcriptome analysis of reprogrammed neurons in vivo . These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6‐OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV‐dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease. Synopsis GABAergic neurons generated by CRISPR‐mediated direct reprogramming of striatal astrocytes rescue voluntary motor behavior in a toxin‐induced murine model for Parkinson's disease, suggesting a novel intervention strategy beyond the restoration of dopamine levels. A novel CRISPRa mouse line dCAM is developed for the conditional induction of endogenous target genes. An AAV‐based split‐dCas9‐activator system is established for translational applications of CRISPRa. Direct reprogramming of murine striatal astrocytes using the factor combination Ascl1 , Lmx1a , and Nr4a2 results in induced GABAergic neurons in vivo . Induced GABAergic neurons are capable of ameliorating specific motor symptoms of Parkinson's disease. Graphical Abstract GABAergic neurons generated by CRISPR‐mediated direct reprogramming of striatal astrocytes rescue voluntary motor behavior in a toxin‐induced murine model for Parkinson's disease, suggesting a novel intervention strategy beyond the restoration of dopamine levels.

Understanding how gene expression impacts plant development and physiology is important for crop engineering. Programmable transcriptional activators (PTAs), including CRISPR-Cas activators, have relied on a limited number of transcriptional activation domains (ADs) (Casas-Mollano et al., 2020; Lowder et al., 2018; Pan et al., 2021; Papikian et al., 2019). Usually, the VP64 domain, derived from herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. In dCas9-based PTAs, binding to a target DNA sequence is afforded by the dCas9-sgRNA ribonucleoprotein. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived AD.

Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological tools that go far beyond its original function as “genetic scissors”. One of these further developments of the CRISPR-Cas system allows genes to be activated in a targeted and efficient manner. These gene-activating CRISPR-Cas modules (CRISPRa) are based on a programmable recruitment of transcription factors to specific loci and offer several key advantages that make them particularly attractive for therapeutic applications. These advantages include inter alia low off-target effects, independence of the target gene size as well as the potential to develop gene- and mutation-independent therapeutic strategies. Herein, I will give an overview on the currently available CRISPRa modules and discuss recent developments, future potentials and limitations of this approach with a focus on therapeutic applications and in vivo delivery.

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example, we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes ‘Organoid Easytag’, our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly targeted cells. Our toolbox also incorporates conditional gene knockdown or overexpression using tightly inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many ongoing descriptive studies, such as the Human Cell Atlas Project.

Bone tissue regeneration and fracture healing remain a significant challenge for physicians, with nonunion failures occurring in an estimated 5%–10% of bone-healing treatments. The autologous bone graft has long been the gold standard of treatment. However, these procedures suffer from persistent donor-site morbidity and extended surgery times, while still having high revision and nonunion failure rates. Cell therapies and tissue engineering strategies utilizing stem cells have been considered as promising alternatives to autologous bone grafts. Here, we explore the concept of using CRISPR-activation (CRISPRa) as a cell-engineering tool to drive osteogenesis without exogenous growth factors. We present a genome-wide CRISPRa screen in adipose-derived stem cells (ASCs) to identify upregulation targets that drive osteogenesis. Top targets from the screen, SPRED2 and ATXN7L3B, demonstrated significant increases in alkaline phosphatase activity and mineralization in monolayer and 3D culture. These results are the first evidence of these genes as osteogenic targets in ASCs.