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Found: 26 179
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SuperFi-Cas9: High Fidelity Meets High Activity.
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Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing.
The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.
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Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective.
In recent years, next-generation sequencing (NGS) and contemporary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have revolutionized the life sciences and the field of plant virology. Both these technologies offer an unparalleled platform for sequencing and deciphering viral metagenomes promptly. Over the past two decades, NGS technologies have improved enormously and have impacted plant virology. NGS has enabled the detection of plant viruses that were previously undetectable by conventional approaches, such as quarantine and archeological plant samples, and has helped to track the evolutionary footprints of viral pathogens. The CRISPR-Cas-based genome editing (GE) and detection techniques have enabled the development of effective approaches to virus resistance. Different versions of CRISPR-Cas have been employed to successfully confer resistance against diverse plant viruses by directly targeting the virus genome or indirectly editing certain host susceptibility factors. Applications of CRISPR-Cas systems include targeted insertion and/or deletion, site-directed mutagenesis, induction/expression/repression of the gene(s), epigenome re-modeling, and SNPs detection. The CRISPR-Cas toolbox has been equipped with precision GE tools to engineer the target genome with and without double-stranded (ds) breaks or donor templates. This technique has also enabled the generation of transgene-free genetically engineered plants, DNA repair, base substitution, prime editing, detection of small molecules, and biosensing in plant virology. This review discusses the utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology.
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Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don).
BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.
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Tumor targeted genome editing mediated by a multi-functional gene vector for regulating cell behaviors.
For effective regulation of cell behaviors and prevention of tumor development by genome editing, we constructed multi-functional self-assembled nanoparticles based on natural polymers to deliver CRISPR-Cas9 plasmid to tumorous cells. The CRISPR based gene editing plasmid to knockout CDK11 gene was complexed with protamine sulfate, and then the complex was decorated by a multi-functional outer layer composed of an endosomolytic peptide (KALA) and aptamer AS1411 incorporated carboxymethyl chitosan. The resultant multi-functional nanoparticles, which exhibit significantly enhanced delivery efficiency, can specifically deliver the plasmid into tumor cell nuclei owing to the favorable effects of KALA in cellular uptake and endosomal escape, together with the cancer cell and cell nucleus targeting capability of AS1411 ligands. The genome editing mediated by the nanoparticles leads to a dramatic decrease (>75%) in CDK11 expression, which results in further modulation of cancer cells with significant down-regulation of the proteins (MMP-9 and VEGF) involved in tumor development and metastasis as well as up-regulation of the tumor suppressor protein p53. More importantly, the detection of immune-related proteins after genome editing shows that the significantly enhanced Fas, CD80, MICA, MICB, and HLA-1 expression and decreased CD47 and MUC1 expression, indicating the genome editing is favorable for reversal of tumor-induced immunosuppression and prevention of tumor development.
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Developing small Cas9 hybrids using molecular modeling.
The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington's disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.
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mTREX2 co-expression promotes large deletions in CRISPR/Cas12a editing of the highly polyploid sugarcane genome.
The LbCas12ai-mTREX2 system enhances large deletions, and the LbCas12ai-RRV enables efficient editing of lw1 in sugarcane. Sugarcane (Saccharum spp. hybrids) is a major feedstock for biofuel production and the primary source of global table sugar; however, its highly polyploid and complex genome constrains the effective deployment of functional genomics and precision breeding strategies. While CRISPR/Cas9-mediated genome editing has been reported in sugarcane, Cas12a-based editing remains unexplored despite its advantages in targeting AT-rich genomic regions. In this study, we report efficient CRISPR/Cas12a-mediated targeted mutagenesis of the lw1 gene in sugarcane. The lemon white1 (lw1) is a key regulator of chlorophyll and carotenoid biosynthesis, and targeted lw1 mutagenesis results in a visible phenotype. We compared the editing performance of intron-containing LbCas12a-R (LbCas12ai-R) and LbCas12a-RRV (LbCas12ai-RRV) variants and assessed the impact of Mus musculus Three prime repair exonuclease 2 (mTREX2) co-expression on editing efficiency and deletion profiles. Editing efficiencies ranged from 31 to 85%, where LbCas12ai-RRV consistently outperformed LbCas12ai-R in both overall editing efficiency and allelic editing efficiency. Co-expression of the mTREX2 shifted the mutation spectrum toward larger deletions, increasing the frequency of moderate-to-large deletions. The ≥ 51 bp deletion class increased ~7-fold in both variants. Capillary electrophoresis-based genotyping provided a cost-effective, high-throughput alternative to next-generation sequencing (NGS) and accurately predicted allelic editing efficiency and indel size. This first report of CRISPR/Cas12a-mediated genome editing in sugarcane provides a framework for future engineering of AT-rich cis-regulatory elements in the polyploid sugarcane genome.
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Advances in genome editing for improved animal breeding: A review.
Since centuries, the traits for production and disease resistance are being targeted while improving the genetic merit of domestic animals, using conventional breeding programs such as inbreeding, outbreeding, or introduction of marker-assisted selection. The arrival of new scientific concepts, such as cloning and genome engineering, has added a new and promising research dimension to the existing animal breeding programs. Development of genome editing technologies such as transcription activator-like effector nuclease, zinc finger nuclease, and clustered regularly interspaced short palindromic repeats systems begun a fresh era of genome editing, through which any change in the genome, including specific DNA sequence or indels, can be made with unprecedented precision and specificity. Furthermore, it offers an opportunity of intensification in the frequency of desirable alleles in an animal population through gene-edited individuals more rapidly than conventional breeding. The specific research is evolving swiftly with a focus on improvement of economically important animal species or their traits all of which form an important subject of this review. It also discusses the hurdles to commercialization of these techniques despite several patent applications owing to the ambiguous legal status of genome-editing methods on account of their disputed classification. Nonetheless, barring ethical concerns gene-editing entailing economically important genes offers a tremendous potential for breeding animals with desirable traits.
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Heterologous Expression of Candida antarctica Lipase B in Aspergillus niger Using CRISPR/Cas9-mediated Multi-Gene Editing.
Aspergillus niger, a filamentous fungus, is known as a cell factory due to its ability to produce large amounts of organic acids and industrial enzymes. Lipase B from Candida antarctica (CALB) is one of the most widely used lipases in industrial applications, including oil processing, papermaking, food, pharmaceuticals, and personal care products. In this study, the CRISPR/Cas9 technique was employed to knock out the pyrG and kusA genes in A. niger. The CALB gene was integrated into the high-production protein gene loci, such as glaA and amyA, to construct a multi-copy CALB production engineered strain. Additionally, the pepA, aglU, and bglA genes were deleted, which minimized the background level of secreted proteins in A. niger and increased the production of CALB. After two rounds of gene editing, the A. niger with multi-copy CALB was created, and the engineered A. niger CCTCC 206047.09 with high CALB yield was isolated. After 120 h of liquid fermentation, the lipase activity reached 17.84 U/mL and the protein yield reached 10.21 mg/mL. In summary, an engineered A. niger strain with high lipase activity was successfully isolated by employing a CRISPR/Cas9 system to integrate CALB into high-expression loci, while simultaneously knocking out the host's highly expressed protein genes. These results provide an effective strategy for the high expression of both heterologous and homologous enzymes in A. niger.
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RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium.
Members of the genus Clostridium represent a diverse assemblage of species exhibiting both medical and industrial importance. Deriving both a greater understanding of their biology, while at the same time enhancing their exploitable properties, requires effective genome editing tools. Here, we demonstrate the first implementation in the genus of theophylline-dependent, synthetic riboswitches exhibiting a full set of dynamic ranges, also suitable for applications where tight control of gene expression is required. Their utility was highlighted by generating a novel riboswitch-based editing tool-RiboCas-that overcomes the main obstacles associated with CRISPR/Cas9 systems, including low transformation efficiencies and excessive Cas9 toxicity. The universal nature of the tool was established by obtaining chromosomal modifications in C. pasteurianum, C. difficile, and C. sporogenes, as well as by carrying out the first reported example of CRISPR-targeted gene disruption in C. botulinum. The high efficiency (100% mutant generation) and ease of application of RiboCas make it suitable for use in a diverse range of microorganisms.
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