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Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.
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Therapeutic Application of Genome Editing Technologies in Viral Diseases.
Viral infections can be fatal and consequently, they are a serious threat to human health. Therefore, the development of vaccines and appropriate antiviral therapeutic agents is essential. Depending on the virus, it can cause an acute or a chronic infection. The characteristics of viruses can act as inhibiting factors for the development of appropriate treatment methods. Genome editing technology, including the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), is a technology that can directly target and modify genomic sequences in almost all eukaryotic cells. The development of this technology has greatly expanded its applicability in life science research and gene therapy development. Research on the use of this technology to develop therapeutics for viral diseases is being conducted for various purposes, such as eliminating latent infections or providing resistance to new infections. In this review, we will look at the current status of the development of viral therapeutic agents using genome editing technology and discuss how this technology can be used as a new treatment approach for viral diseases.
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Developing a CRISPR System in Nongenetic Model Polyploids.
The genetic consequences following polyploidy (i.e., whole-genome duplication; WGD) vary greatly across organisms and through time since polyploidization. At the gene level in allopolyploids, changes include loss/retention of both parental gene copies, function/expression divergence between the two parental copies, and silencing of one parental copy. Functional studies of genes with different retention patterns contribute to a better understanding of the genetic factors underlying the success of polyploids. Most research on gene functions to date focuses on a few well-established genetic models or crops. However, many species that best exemplify the polyploidy process are nongenetic models; the lack of an efficient genome editing system hinders functional studies in these systems. In this chapter, we discuss the considerations of developing CRISPR, a robust and efficient genome editing system, in polyploid plants that are not genetic models. We use diploid and polyploid Tragopogon (Asteraceae) as examples of a well-studied evolutionary model system for which abundant genetic and genomic resources are lacking. Using this system, we provide our protocols for sgRNA design, plasmid construction, a useful protoplast transient assay, and a plant transformation method we developed for this system. We also provide suggestions for possible modifications to these protocols to help promote successful application to other non-models. With the rapid applications of CRISPR in plant sciences, the broad adaptation of CRISPR in studies of the evolutionary significance of WGD holds enormous potential. We hope our studies and methods developed for polyploid Tragopogon will provide a guideline for establishing a CRISPR system in other nongenetic model polyploids of evolutionary or other interest.
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Selection of chromosomal DNA libraries using a multiplex CRISPR system.
The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
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Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage.
Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.
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sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.
Although the CRISPR/Cas9/sgRNA system efficiently cleaves intracellular DNA at desired target sites, major concerns remain on potential "off-target" cleavage that may occur throughout the whole genome. In order to improve CRISPR-Cas9 specificity for targeted genome editing and transcriptional control, we describe a bioinformatics tool "sgRNAcas9", which is a software package developed for fast design of CRISPR sgRNA with minimized off-target effects. This package consists of programs to perform a search for CRISPR target sites (protospacers) with user-defined parameters, predict genome-wide Cas9 potential off-target cleavage sites (POT), classify the POT into three categories, batch-design oligonucleotides for constructing 20-nt (nucleotides) or truncated sgRNA expression vectors, extract desired length nucleotide sequences flanking the on- or off-target cleavage sites for designing PCR primer pairs to validate the mutations by T7E1 cleavage assay. Importantly, by identifying potential off-target sites in silico, the sgRNAcas9 allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of sgRNA for genome editing applications. sgRNAcas9 software package is publicly available at BiooTools website (www.biootools.com) under the terms of the GNU General Public License.
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Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using the CRISPR/Cas9 and piggyBac transposon system.
BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use. OBJECTIVE: In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation. METHODS: For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression. RESULTS: After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their "stemness". When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored. CONCLUSIONS: Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.
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Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.
The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.
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CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond.
Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics.
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Harnessing diverse tRNAs and AI-guided mining for compact and efficient plant multiplex genome editing.
The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.
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