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Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.
Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.
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Expanding the Range of CRISPR/Cas9 Genome Editing in Rice.
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Switching the activity of Cas12a using guide RNA strand displacement circuits.
The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.
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Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.
BACKGROUND: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results. RESULTS: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission. CONCLUSIONS: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.
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CRISPR-Cas: a game-changer in vaccine development and the fight against viral infections.
Understanding the mechanisms of virus-host interactions is crucial for advancing our knowledge of viral pathogenesis and developing effective antiviral strategies. As novel, highly virulent pathogens continue to emerge, overcoming the challenges in virus-host biology and infection control is more important than ever. The CRISPR-Cas system has revolutionized genetic engineering, offering a precise and efficient application in synthetic biology. It is also invaluable in virology research, diagnostics, and antiviral therapeutics. In this review, we explore the latest advances in CRISPR-Cas-based approaches to elucidate virus-host interactions, focusing on the identification of key receptors and host factors essential for virus infection. Furthermore, we discuss the potential of the CRISPR-Cas system as an antiviral strategy for tackling newly emerging and re-emerging viruses. Finally, we discuss the potential of CRISPR-Cas-based gene editing for the development of innovative viral vaccines.
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DGAT2 stability is increased in response to DGAT1 inhibition in gene edited HepG2 cells.
In eukaryotic organisms, two unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, catalyze the final step of the triacylglycerol biosynthetic pathway. Both enzymes are highly expressed in lipogenic tissues, such as adipose tissue, small intestine and the liver. DGAT2 has a prominent role in hepatocyte lipid metabolism synthesizing triacylglycerols that are utilized for very low-density lipoprotein assembly. However, due to the lack of useful antibodies to detect endogenous DGAT2 protein, it has been difficult to determine how this enzyme functions at the cellular level. We have unsuccessfully tested many commercial antibodies as well as our own "in-house" antibodies. There is currently no evidence that DGAT2 undergoes processing such that antigenic epitopes to these antibodies are removed. As an alternative, many studies have utilized epitope tagged versions of DGAT2 overexpressed in cells. These approaches can provide valuable information about a protein, but can be subject to artifacts, such as mislocalization, misregulation, protein aggregation and abnormal protein-protein interactions. In this study, we used gene editing with CRISPR/Cas9 to add three consecutive FLAG epitopes to the C-terminus of endogenous DGAT2 in HepG2 cells. HepG2 cells, derived from a human hepatocellular carcinoma, have been routinely used as a cell model to study human hepatocyte lipid and lipoprotein metabolism. Using this system allowed us to successfully detect DGAT2 expressed from its endogenous locus in HepG2 cells by immunoblotting with anti-FLAG antibodies.
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When Less Is More: Targeting the Myostatin Gene in Livestock for Augmenting Meat Production.
How to increase meat production is one of the main questions in animal breeding. Selection for improved body weight has been made and, due to recent genomic advances, naturally occurring variants that are responsible for controlling economically relevant phenotypes have been revealed. The myostatin (MSTN) gene, a superstar gene in animal breeding, was discovered as a negative controller of muscle mass. In some livestock species, natural mutations in the MSTN gene could generate the agriculturally desirable double-muscling phenotype. However, some other livestock species or breeds lack these desirable variants. Genetic modification, particularly gene editing, offers an unprecedented opportunity to induce or mimic naturally occurring mutations in livestock genomes. To date, various MSTN-edited livestock species have been generated using different gene modification tools. These MSTN gene-edited models have higher growth rates and increased muscle mass, suggesting the high potential of utilizing MSTN gene editing in animal breeding. Additionally, post-editing investigations in most livestock species support the favorable influence of targeting the MSTN gene on meat quantity and quality. In this Review, we provide a collective discussion on targeting the MSTN gene in livestock to further encourage its utilization opportunities. It is expected that, shortly, MSTN gene-edited livestock will be commercialized, and MSTN-edited meat will be on the tables of ordinary customers.
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Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery.
Non-viral polymeric vectors with good biocompatibility have been recently explored as delivery systems for clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases. In this review, based on current limitations and critical barriers, we summarize the advantages of stimulus-responsive polymeric delivery vectors (i.e., pH, redox, or enzymes) towards controllable CRISPR/Cas9 genome editing system delivery as well as the advances in using stimulus-responsive CRISPR/Cas9 polymeric carriers towards cancer treatment. Last but not least, the key challenges and promising development strategies of stimulus-responsive polymeric vector designs for CRISPR/Cas9 systems will also be discussed.
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Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1.
CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.
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Revolutionizing personalized cancer treatment: the synergy of next-generation sequencing and CRISPR/Cas9.
In the context of cancer heterogeneity, the synergistic action of next-generation sequencing (NGS) and CRISPR/Cas9 plays a promising role in the personalized treatment of cancer. NGS enables high-throughput genomic profiling of tumors and pinpoints specific mutations that primarily lead to cancer. Oncologists use this information obtained from NGS in the form of DNA profiling or RNA analysis to tailor precision strategies based on an individual's unique molecular signature. Furthermore, the CRISPR technique enables precise editing of cancer-specific mutations, allowing targeted gene modifications. Harnessing the potential insights of NGS and CRISPR/Cas9 heralds a remarkable frontier in cancer therapeutics with unprecedented precision, effectiveness and minimal off-target effects.
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