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Gene Editing in Potato Using CRISPR-Cas9 Technology.
Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis.
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Functional Analysis of M-Locus Protein Kinase Revealed a Novel Regulatory Mechanism of Self-Incompatibility in Brassica napus L.
Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.
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PrimeNet: rational design of Prime editing pegRNAs by deep learning.
The rapid development of gene editing technology has revolutionized life science research and biotechnology applications. Prime editing, a precise gene editing tool, has shown promise in various applications, including disease research and therapeutic interventions. However, its suboptimal editing efficiency for extensive fragments and lack of predictive models have hindered its widespread adoption. Existing models exhibit low prediction accuracy and limitations, such as neglecting epigenetic factors that impact gene editing effects. To address these challenges, we developed PrimeNet, a novel prediction model that integrates significant epigenetic factors, including chromatin accessibility and DNA methylation. By incorporating data from multiple cell lines and introducing multiscale convolution and attention mechanisms, PrimeNet enhances the accuracy of predictions and generalization performance. Our results show that PrimeNet achieves a Spearman correlation coefficient of 0.94 and 0.82 on two datasets originated from HEK293T and K562 cell lines, respectively, outperforming existing models. This novel model has the potential to guide experimental design, enhance the success rate of gene editing, and reduce unnecessary experimental costs, thereby advancing the application of gene editing technology in genetic disease treatment and related fields.
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Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes.
Advances in gene-editing technology enable efficient, targeted ex vivo engineering of different cell types, which offer a potential therapeutic platform for most challenging disease areas. CRISPR-Cas9 is a widely used gene-editing tool in therapeutic applications. The quality of gene-editing reagents (i.e., Cas9 nuclease, single guide (sg)RNA) is associated with the final cellular product quality as they can impact the gene-editing accuracy and efficiency. To assess the impact of the quality of Cas9 protein and sgRNA in the formation of a Cas9 ribonucleoprotein (RNP) complex, stability, and functional activities, we developed a size exclusion chromatography method that utilizes multiple detectors and an in vitro DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides new tools to characterize the Cas9 RNP complex formation, stability, and functional activity and provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.
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PreAcrs: a machine learning framework for identifying anti-CRISPR proteins.
BACKGROUND: Anti-CRISPR proteins are potent modulators that inhibit the CRISPR-Cas immunity system and have huge potential in gene editing and gene therapy as a genome-editing tool. Extensive studies have shown that anti-CRISPR proteins are essential for modifying endogenous genes, promoting the RNA-guided binding and cleavage of DNA or RNA substrates. In recent years, identifying and characterizing anti-CRISPR proteins has become a hot and significant research topic in bioinformatics. However, as most anti-CRISPR proteins fall short in sharing similarities to those currently known, traditional screening methods are time-consuming and inefficient. Machine learning methods could fill this gap with powerful predictive capability and provide a new perspective for anti-CRISPR protein identification. RESULTS: Here, we present a novel machine learning ensemble predictor, called PreAcrs, to identify anti-CRISPR proteins from protein sequences directly. Three features and eight different machine learning algorithms were used to train PreAcrs. PreAcrs outperformed other existing methods and significantly improved the prediction accuracy for identifying anti-CRISPR proteins. CONCLUSIONS: In summary, the PreAcrs predictor achieved a competitive performance for predicting new anti-CRISPR proteins in terms of accuracy and robustness. We anticipate PreAcrs will be a valuable tool for researchers to speed up the research process. The source code is available at: https://github.com/Lyn-666/anti_CRISPR.git .
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Can Designer Indels Be Tailored by Gene Editing?: Can Indels Be Customized?
Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical- or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w.
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Expanding the RNA-editing toolbox.
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CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.
CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.
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CRISPR-Mediated Viral Gene Knockout to Investigate Viral Evasion of Antiviral Innate Immunity.
The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.
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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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