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Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.
Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.
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Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation.
Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.IMPORTANCE Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.
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Phenotypic novelty by CRISPR in plants.
Genome editing by CRISPR is now routinely used in plant biology for unravelling gene functions and improving agronomical traits. CRISPR opens up the possibility of genome manipulations which would have been unthinkable a few years ago. In this perspective, we discuss and suggest CRISPR-mediated approaches for steering plant development, also highlighting potential challenges.
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Fostering a prevention mindset for responsible gene editing.
In this special issue, we solicited three commentators to discuss issues specific to the responsible conduct of research as it relates to the birth of gene-edited children. We explore the ethics of prevention and its ties to the responsible conduct of gene-editing research to introduce three commentaries addressing: (1) the relevance of cultural and regulatory context in China, (2) how to actualize calls for greater public engagement, and (3) where we might improve graduate education of genomic researchers.
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An ATMT-CRISPR/Cas9 system for genome editing in Monascus purpureus.
Monascus purpureus is a filamentous fungus of significant economic value in the food and pharmaceutical industries, capable of producing a diverse array of secondary metabolites. Although CRISPR/Cas9 systems have been extensively utilized in filamentous fungi, predominantly employing protoplasts as recipients, the genetic manipulation of M. purpureus remains challenging due to the inherent difficulties associated with protoplast preparation. In addition, it has been reported that the Cas9 protein may demonstrate toxicity to cells. To overcome this limitation, this study developed a CRISPR/Cas9 gene-editing system based on Agrobacterium tumefaciens -mediated transformation (ATMT). This system utilizes M. purpureus spores as recipients and employs a homologous recombination strategy to achieve stable, site-specific integration of the Cas9 expression cassette into non-coding regions of the host genome, thereby avoiding the complexity of protoplast preparation and the uncertainty of random integration events. System evaluations indicate that the stable expression of Cas9 protein has no significant adverse effects on the nutritional growth, reproductive development, or characteristic pigment biosynthesis of M. purpureus. The system demonstrated high efficacy in single-gene editing, achieving a knockout efficiency of 74% for the key pigment biosynthetic pathway gene pksPT. Although efficiency decreased when performing double-gene and triple-gene editing (4.8% and 1.7%, respectively), this study successfully validated the system’s potential for multi-gene genome engineering. The ATMT-CRISPR/Cas9 system established herein demonstrates the feasibility of genetic manipulation of M. purpureus, providing a methodological foundation with the potential to facilitating functional genomic studies and the targeted regulation of secondary metabolism in this industrially important fungus.
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Revolutionizing molecular plant breeding through genetic engineering of in vivo haploid induction genes.
Haploid and doubled haploid plants serve critical functions in plant genetics and breeding studies, as they enable the production of genetically uniform lines (pure lines) and thereby accelerate the development of new cultivars. Nowadays, various key genes regulating haploid induction have been found in maize and other plant species (e.g. Arabidopsis, rice, and wheat). These genes offer insights into the mechanisms underlying in vivo haploid induction and provide targets for genetic manipulation. Recent advancements in gene editing and genetic engineering technologies, such as CRISPR/Cas9, have revolutionized our ability to understand gene functions and precisely modify genomes. By leveraging these technologies, scientists can introduce targeted genetic changes, optimize the haploid induction process, and broaden the range of species amenable to haploid production. The application of gene editing and genetic engineering in haploid production holds immense promise for various fields, including agriculture and biotechnology. This focused review aims to advance our understanding of haploid induction genes as well as the potential applications of gene editing/genetic engineering technologies in haploid production, paving the way for innovative solutions to pressing challenges in future plant breeding and agriculture.
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Hematopoietic-Stem-Cell-Targeted Gene-Addition and Gene-Editing Strategies for β-hemoglobinopathies.
Sickle cell disease (SCD) is caused by a well-defined point mutation in the β-globin gene and therefore is an optimal target for hematopoietic stem cell (HSC) gene-addition/editing therapy. In HSC gene-addition therapy, a therapeutic β-globin gene is integrated into patient HSCs via lentiviral transduction, resulting in long-term phenotypic correction. State-of-the-art gene-editing technology has made it possible to repair the β-globin mutation in patient HSCs or target genetic loci associated with reactivation of endogenous γ-globin expression. With both approaches showing signs of therapeutic efficacy in patients, we discuss current genetic treatments, challenges, and technical advances in this field.
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A cut above: pair that developed CRISPR earns historic award.
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The Potential of CRISPR/Cas9 in Hematotherapy.
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Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania.
CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in LeishmaniaIMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.
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