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May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells.
In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.
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CRISPR-Cas9 directed genome engineering for enhancing salt stress tolerance in rice.
Crop productivity in rice is harshly limited due to high concentration of salt in the soil. To understand the intricacies of the mechanism it is important to unravel the key pathways operating inside the plant cell. Emerging state-of-the art technologies have provided the tools to discover the key components inside the plant cell for salt tolerance. Among the molecular entities, transcription factors and/or other important components of sensing and signaling cascades have been the attractive targets and the role of NHX and SOS1 transporters amply described. Not only marker assisted programs but also transgenic approaches by using reverse genetic strategies (knockout or knockdown) or overexpression have been extensively used to engineer rice crop. CRISPR/Cas is an attractive paradigm and provides the feasibility for manipulating several genes simultaneously. Here, in this review we highlight some of the molecular entities that could be potentially targeted for generating rice amenable to sustain growth under high salinity conditions by employing CRISPR/Cas. We also try to address key questions for rice salt stress tolerance other than what is already known.
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Creation of colorless transparent tilapia using CRISPR/Cas9 mediated multi-gene mutation.
Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form tyrb;csf1ra double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of pnp4a and tfec using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in pnp4a mutants but not in tfec mutants in the later stage. In addition, we also found that tfec mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of tyrb;csf1ra;pnp4a (named amber) and tyrb;csf1ra;tfec (named ruby), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.
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Structure-activity relationships study on inhibition of CRISPR-Cas9 by glycosaminoglycans.
The CRISPR-Cas9 system is a revolutionary genome editing system known for its precision, simplicity and efficiency, playing a crucial role in gene-editing. It has enabled applications ranging from biomedicine to agriculture. However, the uncontrollable activity of Cas9 has raised significant safety concerns in clinical settings, limiting its broader application. Consequently, regulating CRISPR-Cas9 activity holds substantial promise for enhancing the safety and efficacy of gene-editing technologies. In this study, we demonstrated that sulfated glycosaminoglycans (GAGs) exhibit inhibitory effects of Cas9. Specifically, both chondroitin sulfate (CS) and heparin (HP) can inhibit CRISPR/Cas9 activity, with heparin showing a stronger inhibitory effect that correlates positively with its concentration. Molecular dynamics simulations indicate that sulfated heparin residues might inhibit Cas9 function by binding to essential DNA-binding sites, which are crucial for functional interactions, potentially impairing activity. Additionally, higher molecular weight GAGs exhibit enhanced inhibitory effects under the same sulphation. Notably, the sulfation site also influenced activity. C6-sulfation of chondroitin sulfate is more favorable for Cas9 inhibition, and N-sulfation of heparin enhances its inhibitory effect on Cas9 activity. These findings provide valuable insights into the development of carbohydrate-based inhibitors for CRISPR-Cas9, offering a foundation for further exploration in this field.
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In vivo promoter engineering in plants: Are we ready?
Engineering plant promoter sequence for optimal expression of a gene has been a long standing goal for plant scientists. In recent times, Sequence Specific Nucleases (SSNs) like CRISPR/Cas9 are enabling researchers to achieve this goal, in vivo in the genome. It is well known that SSNs have met with unprecedented success in rapid transgene free crop improvement largely by targeting the coding sequence. Here, we discuss the strategies being employed by plant scientists in targeting SSNs to non-coding promoter regions/Cis Regulatory Elements (CRE). We collectively refer all such endeavors as in vivo promoter engineering (IPE). We further classify the IPE efforts into CRE addition, CRE deletion/disruption, promoter swap/insertion and targeted promoter polymorphism. Till date, IPE has proven useful in altering plant architecture in tomato, developing resistance against Xanthomonas sp in rice and citrus, and engineering drought tolerance in maize. However it is quite challenging to achieve predictable changes in gene expression using IPE at this point. In future years, data generated from high throughput techniques to investigate non coding genome may immensely augment the efforts in this direction. As IPE does not involve addition of the transgene for modifying crop traits, it will be relatively more conducive to public acceptance in crop improvement programs.
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Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA-ncRNA Expression Constructs.
CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA-ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA "cargo" domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA-ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA-ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of "best practices" for the design and implementation of CRISPR-Display experiments.
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CNN-XG: A Hybrid Framework for sgRNA On-Target Prediction.
As the third generation gene editing technology, Crispr/Cas9 has a wide range of applications. The success of Crispr depends on the editing of the target gene via a functional complex of sgRNA and Cas9 proteins. Therefore, highly specific and high on-target cleavage efficiency sgRNA can make this process more accurate and efficient. Although there are already many sophisticated machine learning or deep learning models to predict the on-target cleavage efficiency of sgRNA, prediction accuracy remains to be improved. XGBoost is good at classification as the ensemble model could overcome the deficiency of a single classifier to classify, and we would like to improve the prediction efficiency for sgRNA on-target activity by introducing XGBoost into the model. We present a novel machine learning framework which combines a convolutional neural network (CNN) and XGBoost to predict sgRNA on-target knockout efficacy. Our framework, called CNN-XG, is mainly composed of two parts: a feature extractor CNN is used to automatically extract features from sequences and predictor XGBoost is applied to predict features extracted after convolution. Experiments on commonly used datasets show that CNN-XG performed significantly better than other existing frameworks in the predicted classification mode.
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Expanding genome editing scopes with artificial intelligence.
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Inducible CRISPR-Cas9 screening platform to interrogate non-proliferative cellular states.
CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.
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Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases.
Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.
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