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CRISPR-Cas technology opens a new era for the creation of novel maize germplasms.
Maize (Zea mays) is one of the most important food crops in the world with the greatest global production, and contributes to satiating the demands for human food, animal feed, and biofuels. With population growth and deteriorating environment, efficient and innovative breeding strategies to develop maize varieties with high yield and stress resistance are urgently needed to augment global food security and sustainable agriculture. CRISPR-Cas-mediated genome-editing technology (clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated)) has emerged as an effective and powerful tool for plant science and crop improvement, and is likely to accelerate crop breeding in ways dissimilar to crossbreeding and transgenic technologies. In this review, we summarize the current applications and prospects of CRISPR-Cas technology in maize gene-function studies and the generation of new germplasm for increased yield, specialty corns, plant architecture, stress response, haploid induction, and male sterility. Optimization of gene editing and genetic transformation systems for maize is also briefly reviewed. Lastly, the challenges and new opportunities that arise with the use of the CRISPR-Cas technology for maize genetic improvement are discussed.
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Potential of Genome Editing to Improve Aquaculture Breeding and Production.
Aquaculture is the fastest growing food production sector and is rapidly becoming the primary source of seafood for human diets. Selective breeding programs are enabling genetic improvement of production traits, such as disease resistance, but progress is limited by the heritability of the trait and generation interval of the species. New breeding technologies, such as genome editing using CRISPR/Cas9 have the potential to expedite sustainable genetic improvement in aquaculture. Genome editing can rapidly introduce favorable changes to the genome, such as fixing alleles at existing trait loci, creating de novo alleles, or introducing alleles from other strains or species. The high fecundity and external fertilization of most aquaculture species can facilitate genome editing for research and application at a scale that is not possible in farmed terrestrial animals.
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A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing.
The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.
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Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing.
CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5'-NGG-3' for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5'-NAA-3' and 5'-NGAA-3' PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans.
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Genome Editing: The Future Begins!
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[New-generation editing].
Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.
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Generation of zebrafish models by CRISPR /Cas9 genome editing.
The CRISPR /Cas system identified in archaea has been adopted and optimized for genome editing purposes in zebrafish. In vitro transcribed guide RNA and Cas9 mRNA are microinjected into fertilized zebrafish embryos to edit the zebrafish genome. Here, we describe how to design a gRNA, a fast method for in vitro transcription of gRNA from oligonucleotides , microinjection into fertilized zebrafish embryos, and a PCR -based restriction fragment length assay to identify mutations at the gRNA target site.
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Genome centric engineering using ZFNs, TALENs and CRISPR-Cas9 systems for trait improvement and disease control in Animals.
Livestock is an essential life commodity in modern agriculture involving breeding and maintenance. The farming practices have evolved mainly over the last century for commercial outputs, animal welfare, environment friendliness, and public health. Modifying genetic makeup of livestock has been proposed as an effective tool to create farmed animals with characteristics meeting modern farming system goals. The first technique used to produce transgenic farmed animals resulted in random transgene insertion and a low gene transfection rate. Therefore, genome manipulation technologies have been developed to enable efficient gene targeting with a higher accuracy and gene stability. Genome editing (GE) with engineered nucleases-Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) regulates the targeted genetic alterations to facilitate multiple genomic modifications through protein-DNA binding. The application of genome editors indicates usefulness in reproduction, animal models, transgenic animals, and cell lines. Recently, CRISPR/Cas system, an RNA-dependent genome editing tool (GET), is considered one of the most advanced and precise GE techniques for on-target modifications in the mammalian genome by mediating knock-in (KI) and knock-out (KO) of several genes. Lately, CRISPR/Cas9 tool has become the method of choice for genome alterations in livestock species due to its efficiency and specificity. The aim of this review is to discuss the evolution of engineered nucleases and GETs as a powerful tool for genome manipulation with special emphasis on its applications in improving economic traits and conferring resistance to infectious diseases of animals used for food production, by highlighting the recent trends for maintaining sustainable livestock production.
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CRISPR/Cas9 Vector Construction for Gene Knockout.
This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.
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Comparative analysis of single and dual sgRNA CRISPR/Cas9 ribonucleoprotein strategies for efficient Park7 gene editing in primary mouse embryonic fibroblasts.
BACKGROUND: Parkinson's disease (PD) is a progressive neurological condition that predominantly affects dopaminergic neurons in the substantia nigra. Mutations or deletions in the Park7 gene may cause a rare autosomal recessive form of early-onset PD. Generating reliable cellular models with specific Park7 disruption is crucial for elucidating the molecular mechanisms underlying PD pathogenesis. METHODS AND RESULTS: This study developed a plasmid-free CRISPR/Cas9 genome-editing method for targeting the Park7 gene in primary mouse embryonic fibroblasts (MEFs) using single- and dual-sgRNA-mediated ribonucleoprotein (RNP) system. Gene modification through the single sgRNA strategy was confirmed by the detection of indels by T7 endonuclease assay, whereas gene editing by the dual sgRNA strategy was validated through detection of large deletions by junctional PCR and Sanger sequencing. Deep amplicon sequencing showed an overall editing frequency of 50.68% and 24.87% for single-sgRNA and dual-sgRNA editing strategies, respectively. While single-sgRNA editing predominantly generated small insertions and deletions, large deletions constituted the predominant editing outcome in the dual-sgRNA group, accounting for 22.23% of the total reads. Further, Western blot analysis demonstrated a 12.03 ± 7.6% reduction in PARK7 protein expression following single sgRNA transfection, whereas dual sgRNA transfection showed a significant reduction of 54.89 ± 9.1% (p < 0.0001). CONCLUSION: This study demonstrates a plasmid-free CRISPR/Cas9 RNP-based approach for targeted Park7 gene editing in primary MEFs. The results underline the importance of considering both the outcomes of genomic editing and the expression of functional protein when evaluating CRISPR/Cas9 approaches. This established approach sets the basis for the development of Park7-edited cellular models to study molecular mechanisms associated with PD and the subsequent development of disease-relevant models.
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