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A thermostable Cas9-based genome editing system for thermophilic acetogenic bacterium Thermoanaerobacter kivui.
Thermoanaerobacter kivui is a thermophilic acetogenic bacterium capable of thriving at elevated temperatures up to 66°C. It metabolizes carbohydrates such as glucose, mannose, and fructose and can also grow lithotrophically utilizing hydrogen (H2) and carbon dioxide (CO2) or carbon monoxide (CO), with acetate serving as its main product. A simple and efficient genome editing system for T. kivui would not only facilitate the understanding of the physiological function of enzymes involved in energy and carbon metabolism but also enable metabolic engineering. To address this issue, we developed a thermostable Cas9-based genome editing system for targeted gene knockout and gene integration into the T. kivui genome. Gene knockout assays were conducted on the adh gene, responsible for encoding alcohol dehydrogenase, and the ldh gene, encoding lactate dehydrogenase. Furthermore, the adhE gene from Thermoanaerobacter ethanolicus, which encodes a bifunctional aldehyde/alcohol dehydrogenase enzyme, was successfully integrated into the T. kivui genome. As a result, the engineered strain was able to produce ethanol. Following a liquid culturing period with kanamycin sulfate for about 72 hours, the efficiency of gene editing was enhanced, resulting in a ratio of mutants out of all colonies obtained of 90%. The results confirm the validity and efficiency of the thermostable Cas9-based genome editing system in T. kivui for gene editing.IMPORTANCEThermophilic acetogenic microorganisms represent an emerging metabolic engineering platform for the production of various biochemicals from hydrogen and carbon dioxide, or synthesis gas, under conditions of high-temperature fermentation. Gas fermentation has gained significant research interest due to its excellent thermodynamics, economic feasibility, and multisubstrate utilization. However, a major obstacle to the use of thermophilic acetogenic microorganisms as metabolic engineering platforms is the scarcity of genetic tools. This study demonstrates a proof of concept for a thermostable Cas9-based genome editing of the thermophilic acetogenic bacterium T. kivui. The system is an important expansion to the genetic toolbox of T. kivui, enabling a better understanding of key enzyme functions and the construction of cell factories for the biotechnological conversion of carbon dioxide and organic substrates into value-added products.
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Development of an RNA aptamer-assisted CRISPR/Cas9 system for efficiently generating and isolating Cas9-free mutants in plant.
The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.
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Adaptation of bacterial natural single guide RNA (tracr-L) for efficient plant genome editing.
A long tracrRNA (tracr-L), which naturally act as single guide RNA, and its truncated version, Δtracr-L, from S. pyogenes, efficiently induce Cas9-mediated double-strand breaks (DSBs) in plant genomic loci, as demonstrated by in vitro cleavage assay and protoplast transfection. CRISPR-Cas system provides a form of immune memory in prokaryotes and archaea, protecting them against viruses and foreign genetic elements. In Streptococcus pyogenes, this system includes the pre-crRNA along with another non-coding RNA, tracrRNA, which aids in CRISPR-based immunity. In S. pyogenes, two distinct tracrRNAs are produced: a long form (tracr-L) and a short form (tracr-S). The tracr-S regulates crRNA biogenesis and Cas9 cleavage, while tracr-L suppresses CRISPR-Cas expression by targeting the Cas9 promoter to prevent autoimmunity. Deleting 79 nucleotides from tracr-L results in Δtracr-L, which retains similar functionality in gene repression. This study investigates, for the first time, the effectiveness of tracr-L, and Δtracr-L in genome editing within plant systems. In vitro cleavage assays using purified Cas9 and synthesized sgRNAs targeting the Cas9 gene, OsPDS, and the OsSWEET11 promoter revealed that across all target sites, tracr-S demonstrated the highest cleavage efficiency compared to tracr-L and Δtracr-L. For in vivo genome editing, we transfected rice protoplasts with tracr-L, Δtracr-L, and tracr-S, targeting three rice genes: OsPDS, OsSPL14, and the promoter of OsSWEET14. Amplicon deep sequencing revealed various types of indels at the target regions across all three tracrRNA versions, indicating comparable levels of efficiency. This study establishes the utility of both the long-form tracrRNA (tracr-L) and its truncated variant (Δtracr-L) in eukaryote genome editing. These two new forms of tracrRNA provide proof of concept and expand the CRISPR-Cas toolkit for plant genome editing applications, and for eukaryotes more broadly.
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CRISPRroots: on- and off-target assessment of RNA-seq data in CRISPR-Cas9 edited cells.
The CRISPR-Cas9 genome editing tool is used to study genomic variants and gene knockouts, and can be combined with transcriptomic analyses to measure the effects of such alterations on gene expression. But how can one be sure that differential gene expression is due to a successful intended edit and not to an off-target event, without performing an often resource-demanding genome-wide sequencing of the edited cell or strain? To address this question we developed CRISPRroots: CRISPR-Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites. Our method combines Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells to discover potential off-targets. Applied on seven public datasets, CRISPRroots identified critical off-target candidates that were overlooked in all of the corresponding previous studies. CRISPRroots is available via https://rth.dk/resources/crispr.
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Experimental strategy for characterization of novel TnpB orthologs.
TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.
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Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast.
CRISPR is a versatile technology for genomic editing and regulation, but the expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and expressed from a single transcriptional unit. We demonstrate that these gRNA arrays can stably regulate gene expression in a synergistic manner via dCas9-mediated repression. This approach expands the number of gRNAs that can be expressed in this model organism and may enable the versatile editing or transcriptional regulation of a greater number of genes in vivo.
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Communicating Biotech Advances: Fiction versus Reality.
Bioscience novels use selected technologies of genetic engineering and synthetic biology to create entertaining stories. These novels are usually based on scientific knowledge, but they may arouse public concerns about technology and drive public reluctance to accept innovative technologies. The scientific community must adopt more efficient communication and transparency.
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Advances and challenges in genetic technologies to produce single-sex litters.
There is currently a requirement for single-sex litters for many applications, including agriculture, pest control, and reducing animal culling in line with the 3Rs principles: Reduction, Replacement, and Refinement. The advent of CRISPR/Cas9 genome editing presents a new opportunity with which to potentially generate all-female or all-male litters. We review some of the historical nongenetic strategies employed to generate single-sex litters and investigate how genetic and genome editing techniques are currently being used to produce all-male or all-female progeny. Lastly, we speculate on future technologies for generating single-sex litters and the possible associated challenges.
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CRISPR babies: a view from the centre of the storm.
Towards the end of November 2018, news broke that the Chinese researcher He Jiankui had created the world's first genome-edited babies. This came shortly before the start of the Second International Summit on Human Genome Editing, where researchers, ethicists and others concerned with regulation, social issues and public engagement from around the world gathered to discuss the latest advances in the field. In this Spotlight, I provide my perspective on the events that occurred shortly prior to and at the summit, where He Jiankui gave an account of his activities. I also discuss what was wrong with his approach and how, after more research and with appropriate regulation, clinical applications of germline genome editing in humans may be justifiable.
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FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).
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