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Genome-wide specificity of plant genome editing by both CRISPR-Cas9 and TALEN.
CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.
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Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system.
Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.
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What Is Prudent Governance of Human Genome Editing?
CRISPR technology has made questions about how best to regulate human genome editing immediately relevant. A sound and ethical governance structure for human genome editing is necessary, as the consequences of this new technology are far-reaching and profound. Because there are currently many risks associated with genome editing technology, the extent of which are unknown, regulatory prudence is ideal. When considering how best to create a prudent governance scheme, we can look to 2 guiding examples: the Asilomar conference of 1975 and the German Ethics Council guidelines for human germline intervention. Both models offer a path towards prudent regulation in the face of unknown and significant risks.
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A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in Acremonium chrysogenum.
Acremonium chrysogenum is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems for A. chrysogenum employ heterologous promoters, including Aspergillus nidulans PgpdA or Aspergillus fumigatus AfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload. Here, we developed a simplified and highly efficient CRISPR/Cas9 genome editing system using the endogenous 5S rRNA promoter for sgRNA transcription in A. chrysogenum. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting the sorB gene. Furthermore, this platform enabled 100% single gene deletion and efficient large-scale chromosomal deletion, up to 66.17 kb within the sorbicillinoid biosynthetic gene cluster, without donor DNA. To our knowledge, this represents the largest chromosomal deletion reported in A. chrysogenum to date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at the kusA locus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in A. chrysogenum.
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Sequence-independent optical regulation of CRISPR/Cas editing using star-shaped crRNA dendrimers.
Precise spatiotemporal control of CRISPR/Cas editing is vital for studying dynamic processes and ensuring therapeutic safety. We present a single-site photolabile crRNA dendrimer platform enabling robust, sequence-independent optical regulation of Cas9 and Cas12a. This simple, universal strategy achieves rapid OFF-to-ON control with minimal leakage, advancing programmable, light-responsive genome editing for biomedical applications.
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Mucopolysaccharidoses type I gene therapy.
Mucopolysaccharidoses type I (MPS I) is an inherited metabolic disease characterized by a malfunction of the α-l-iduronidase (IDUA) enzyme leading to the storage of glycosaminoglycans in the lysosomes. This disease has longtime been studied as a therapeutic target for those studying gene therapy and many studies have been done using various vectors to deliver the IDUA gene for corrective treatment. Many vectors have difficulties with efficacy and insertional mutagenesis concerns including adeno-associated viral (AAV) vectors. Studies of AAV vectors treating MPS I have seemed promising, but recent deaths in gene therapy clinical trials for other inherited diseases using AAV vectors have left questions about their safety. Additionally, the recent modifications to adenoviral vectors leading them to target the vascular endothelium minimizing the risk of hepatotoxicity could lead to them being a viable option for MPS I gene therapy when coupled with gene editing technologies like CRISPR/Cas9.
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Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9+ T Cells.
Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3+ T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9+CD3+ T cells, CD4+ and CD8+ conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9+CD3+ T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.
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A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast.
CRISPR/Cas9 technology allows accurate, marker-less genome editing. We report a detailed, robust, and streamlined protocol for CRISPR/Cas9 genome editing in Saccharomyces cerevisiae, based on the widely used MoClo-Yeast Toolkit (https://www.addgene.org/kits/moclo-ytk/). This step-by-step protocol guides the reader from sgRNA design to verification of the desired genome editing event and provides preassembled plasmids for cloning the sgRNA(s), making this technology easily accessible to any yeast research group. For complete details on the use and execution of this protocol, please refer to Novarina et al. (2021).
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CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.
With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.
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Advanced Nanotheranostics of CRISPR/Cas for Viral Hepatitis and Hepatocellular Carcinoma.
Liver disease, particularly viral hepatitis and hepatocellular carcinoma (HCC), is a global healthcare burden and leads to more than 2 million deaths per year worldwide. Despite some success in diagnosis and vaccine development, there are still unmet needs to improve diagnostics and therapeutics for viral hepatitis and HCC. The emerging clustered regularly interspaced short palindromic repeat/associated proteins (CRISPR/Cas) technology may open up a unique avenue to tackle these two diseases at the genetic level in a precise manner. Especially, liver is a more accessible organ over others from the delivery point of view, and many advanced strategies applied for nanotheranostics can be adapted in CRISPR-mediated diagnostics or liver gene editing. In this review, the focus is on these two aspects of viral hepatitis and HCC applications. An overview on CRISPR editor development and current progress in clinical trials is first given, followed by highlighting the recent advances integrating the merits of gene editing and nanotheranostics. The promising systems that are used in other applications but may hold potentials in liver gene editing are also discussed. This review concludes with the perspectives on rationally designing the next-generation CRISPR approaches and improving the editing performance.
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