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CRISPR/Cas-mediated editing of cis-regulatory elements for crop improvement.
To improve future agricultural production, major technological advances are required to increase crop production and yield. Targeting the coding region of genes via the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has been well established and has enabled the rapid generation of transgene-free plants, which can lead to crop improvement. The emergence of the CRISPR/Cas system has also enabled scientists to achieve cis-regulatory element (CRE) editing and, consequently, engineering endogenous critical CREs to modulate the expression of target genes. Recent genome-wide association studies have identified the domestication of natural CRE variants to regulate complex agronomic quantitative traits and have allowed for their engineering via the CRISPR/Cas system. Although engineering plant CREs can be advantageous to drive gene expression, there are still many limitations to its practical application. Here, we review the current progress in CRE editing and propose future strategies to effectively target CREs for transcriptional regulation for crop improvement.
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CRISPR/Cas9-mediated genome editing in Ganoderma lucidum: recent advances and biotechnological opportunities.
Ganoderma lucidum is a well-known traditional medicinal mushroom that has attracted considerable attention due to its potential as a promising cell factory for producing high-value bioactive compounds. However, conventional methods for the genetic manipulation of G. lucidum are often time-consuming and labor-intensive, hindering research into the biosynthesis and regulatory mechanisms of its valuable natural products. In recent years, the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system has emerged as a powerful genome editing tool, extensively utilized in life sciences research due to its high efficiency and user-friendliness. This review provides a structured overview of advancements in CRISPR/Cas9-mediated genome editing technology in G. lucidum. We first discuss the development and optimization of the CRISPR system, focusing on the various expression strategies for Cas9 and guide RNA established in G. lucidum. Furthermore, we highlight the application of this system for targeted gene deletion, insertion, and replacement in genome editing, as well as its use in the functional analysis of genes in G. lucidum. In addition, we discuss the limitations and challenges associated with employing CRISPR/Cas9 tools in G. lucidum and provide an outlook on the future development of the CRISPR/Cas9 system and its applications in this organism.
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Application of CRISPR/Cas9 technologies combined with iPSCs in the study and treatment of retinal degenerative diseases.
Retinal degeneration diseases, such as age-related macular degeneration and retinitis pigmentosa, affect millions of people worldwide and are major causes of irreversible blindness. Effective treatments for retinal degeneration, including drug therapy, gene augmentation or transplantation approaches, have been widely investigated. Nevertheless, more research should be dedicated to therapeutic methods to improve future clinical treatments. Recently, with the rapid development of genome-editing technology, gene therapy has become a potentially effective treatment for retinal degeneration diseases. A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a powerful genome-editing tool in ophthalmic studies. The CRISPR/Cas9 system has been widely applied in basic research to develop animal models and gene therapies in vivo. With the ability to self-renew and the potential to differentiate into different types of cells, induced pluripotent stem cells (iPSCs) have already been used as a promising tool for understanding disease pathophysiology and evaluating the effect of drug and gene therapeutics. iPSCs are also a cell source for autologous transplantation. In this review, we compared genome-editing strategies and highlighted the advantages and concerns of the CRISPR/Cas9 system. Moreover, the latest progress and applications of the CRISPR/Cas9 system and its combination with iPSCs for the treatment of retinal degenerative diseases are summarized.
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CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo.
Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (-/-) heterozygous, bi-allelic (-/-) homozygous, and mono-allelic (-/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (-/-) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.
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Spatial control of in vivo CRISPR-Cas9 genome editing via nanomagnets.
The potential of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9)-based therapeutic genome editing is hampered by difficulties in the control of the in vivo activity of CRISPR-Cas9. To minimize any genotoxicity, precise activation of CRISPR-Cas9 in the target tissue is desirable. Here, we show that, by complexing magnetic nanoparticles with recombinant baculoviral vectors (MNP-BVs), CRISPR-Cas9-mediated genome editing can be activated locally in vivo via a magnetic field. The baculoviral vector was chosen for in vivo gene delivery because of its large loading capacity and ability to locally overcome systemic inactivation by the complement system. We demonstrate that a locally applied magnetic field can enhance the cellular entry of MNP-BVs, thereby avoiding baculoviral vector inactivation and causing a transient transgene expression in the target tissue. Because baculoviral vectors are inactivated elsewhere, gene delivery and in vivo genome editing via MNP-BVs are tissue specific.
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A High-Resolution Melting Analysis with an Unlabeled Probe for CRISPR/Cas9-Induced ZBED6 Knockout Pigs Detection.
BACKGROUND: The zinc finger BED-type containing six knockout (ZBED6-KO) pigs were created to improve economic traits by increasing the expression of insulin-like growth factor 2. They were generated by CRISPR/CRISPR-associated protein 9 (Cas9) technology and a single-base deletion of ZBED6 was found. An efficient and rapid method was needed to detect this type of pig. OBJECTIVE: This study aimed to develop a high-resolution melting (HRM) method to detect ZBED6-KO pigs. METHODS: An unlabeled probe and two primers were designed to develop the HRM method. The limit of detection, specificity, and accuracy of the established method were tested by the constructed plasmid and DNA extracts of tissue specimens. RESULTS: The limit of detection by the established method was 102 copies/µL. The HRM method with an unlabeled probe showed good specificity and high accuracy. CONCLUSIONS: The established HRM analysis with an unlabeled probe showed it to be a highly effective, rapid, and reliable method to distinguish ZBED6-KO pigs from wild-type pigs. HIGHLIGHTS: It is the first time that HRM analysis with an unlabeled probe has been used in the detection of genome editing pigs by the CRISPR/Cas9 technology.
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Selection is required for efficient Cas9-mediated genome editing in Fusarium graminearum.
Genome engineering using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nucleases, such as Cas9 (CRISPR-associated protein 9), are revolutionising molecular biology. In this study, we established a Cas9-based genome editing system in Fusarium graminearum, a highly destructive fungal pathogen of cereal crops. Although the molecular toolkit of F. graminearum is well developed compared to other fungi, Cas9-mediated engineering offers a number of potential benefits, such as the ability to create marker free mutants in this species. Here we have used a codon-optimised Cas9 nuclease and dual ribozyme-based expression of a single guide RNA (sgRNA) to induce mutations. Cas9-mediated mutations were identified through a fungicide resistance-based phenotypic screen, which selects for null mutations in the FgOs1 gene encoding an osmosensor histidine kinase. In the absence of selection, however, mutations were identified at very low frequency. Examination of the mutant alleles identified suggests that, a microhomology-mediated end joining (MMEJ) DNA repair pathway is likely to be the predominant process involved in erroneous repairing of Cas9-induced double-stranded breaks in F. graminearum.
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CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding.
Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene(s). In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein (RNP) complex assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding.
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Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing.
CRISPR/Cas9 uses a guide RNA (gRNA) molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.
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Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.
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