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Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice.
Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.
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Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.
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Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.
Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.
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A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.
Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co-delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss-of-function mutants of multiple genes from different gene families.
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Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.
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Epigenetic reprogramming in breast cancer: The role of CRISPR-Cas 9.
Epigenetic alterations are known to be a significant factor in the development and advancement of breast cancer (BC), which continues to be a substantial cause of illness and death in women globally. The emergence of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) technology has fundamentally transformed our capacity to edit the genome with unparalleled accuracy, providing novel opportunities for therapeutic intervention. This review examines the utilization of CRISPR-Cas9 to alter epigenetic landscapes to combat BC. We examine the fundamental processes of CRISPR-Cas9 and its derivatives, including dCas9, in their ability to specifically target DNA methylation and histone alterations. The highlighted review showcases the potential of CRISPR-Cas9 in reactivating silenced tumor suppressor genes and inhibiting oncogenes. In addition, we analyze the incorporation of CRISPR-based epigenetic editing into current medicines, offering valuable knowledge on the use of combination therapies to improve treatment effectiveness and overcome resistance. This review intends to highlight the revolutionary potential of CRISPR-Cas9 in generating targeted, personalized therapeutics for BC by explaining the present advancements and future applications. The incorporation of this state-of-the-art technology with conventional and developing therapies holds the potential to establish more efficient and long-lasting remedies in the battle against BC, ultimately enhancing patient results and rates of survival.
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Cas12a is competitive for gene editing in the malaria parasites.
Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.
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Genome Editing in Translational Medicine: An Inventory.
Genomic mutations are the driving force of biological diversity but they are also the cause of a plethora of human diseases ranging from heritable disorders to neurological pathologies and cancer. For most genetic disorders, there is no curative treatment available to date. The demand for precise, preferably patient-specific, treatment regimen offering cure is naturally high. Genome editing by Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas enables targeted manipulation of genomes, thereby offering the opportunity to treat such diseases. While ethical and regulatory guidelines need to be developed and considered, the prospect of genome editing for curative treatment is certainly exciting. Here, we review the current state of therapeutics based on genome editing techniques. We highlight recent breakthroughs, describe clinical trials employing genome editing-based medicine, discuss the benefits and pitfalls, and take a look into the future of genome editing.
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Viral vectors and extracellular vesicles: innate delivery systems utilized in CRISPR/Cas-mediated cancer therapy.
Gene editing-based therapeutic strategies grant the power to override cell machinery and alter faulty genes contributing to disease development like cancer. Nowadays, the principal tool for gene editing is the clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR/Cas9) system. In order to bring this gene-editing system from the bench to the bedside, a significant hurdle remains, and that is the delivery of CRISPR/Cas to various target cells in vivo and in vitro. The CRISPR-Cas system can be delivered into mammalian cells using various strategies; among all, we have reviewed recent research around two natural gene delivery systems that have been proven to be compatible with human cells. Herein, we have discussed the advantages and limitations of viral vectors, and extracellular vesicles (EVs) in delivering the CRISPR/Cas system for cancer therapy purposes.
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Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.
Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.
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