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Potential of small extracellular vesicles as Cas9 delivery tool: a promising approach for gene editing livestock gametes and embryos.
Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.
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Advancing Precision Medicine: Recent Innovations in Gene Editing Technologies.
The advent of gene editing has significantly advanced the field of medicine, opening new frontiers in the treatment of genetic disorders, cancer, and infectious diseases. Gene editing technology remains a dynamic and promising area of research and development. Recent advancements in protein and RNA engineering within this field have addressed critical issues such as imprecise edits, poor editing efficiency, and off-target effects. Advancements in delivery methods have allowed the achievement of therapeutic or even selection-free gene editing efficiency with reduced toxicity in primary cells, thereby enhancing the safety and efficacy of gene manipulation. This progress paves the way for transformative changes in molecular biology, medicine, and other fields. This review provides a comprehensive overview of the advancements in gene editing techniques, focusing on prime editor proteins and their engineered variants. It also explores alternative systems that expand the toolkit for precise genomic modifications and highlights the potential of these innovations in treating hematological disorders, while also discussing the limitations and challenges that remain.
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Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.
The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.
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Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.
CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.
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Optimizing ErCas12a for efficient gene editing in Arabidopsis thaliana.
The ErCas12a nuclease, also known as MAD7, is part of a CRISPR/Cas system from Eubacterium rectale and distantly related to Cas12a nucleases. As it shares only 31% sequence homology with the commonly used AsCas12a, its intellectual property may not be covered by the granted patent rights for Cas12a nucleases. Thus, ErCas12a became an attractive alternative for practical applications. However, the editing efficiency of ErCas12a is strongly target sequence- and temperature-dependent. Therefore, optimization of the enzyme activity through protein engineering is especially attractive for its application in plants, as they are cultivated at lower temperatures. Based on the knowledge obtained from the optimization of Cas12a nucleases, we opted to improve the gene editing efficiency of ErCas12a by introducing analogous amino acid exchanges. Interestingly, neither of these mutations analogous to those in the enhanced or Ultra versions of AsCas12a resulted in significant editing enhancement of ErCas12a in Arabidopsis thaliana. However, two different mutations, V156R and K172R, in putative alpha helical structures of the enzyme showed a detectable improvement in editing. By combining these two mutations, we obtained an improved ErCas12a (imErCas12a) variant, showing several-fold increase in activity in comparison to the wild-type enzyme in Arabidopsis. This variant yields strong editing efficiencies at 22 °C which could be further increased by raising the cultivation temperature to 28 °C and even enabled editing of formerly inaccessible targets. Additionally, no enhanced off-site activity was detected. Thus, imErCas12a is an economically attractive and efficient alternative to other CRISPR/Cas systems for plant genome engineering.
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ZEB1 and ZEB2 gene editing mediated by CRISPR/Cas9 in A549 cell line.
OBJECTIVES: One of the best approaches for recognition of protein function is the induction of mutations for a gene knockout. In line with this strategy, gene editing tools allow researchers to induce these mutations. Lung cancer is one of the leading causes of death worldwide. ZEB1 and ZEB2 genes are the candidates for this disease. METHODS: The ZEB1 and ZEB2 knockout in the non-small cell lung cancer cell line (A549 cell) was investigated. Purification of recombination plasmids was performed from bacteria and then was transported to the A549 cell line. The deletion of ZEB1 and ZEB2 were examined by PCR. RESULTS: The results demonstrated the mutation and deletion in ZEB1 and ZEB2 genes. Based on the findings of this study, A549 cells were transfected with the vectors carrying the sgRNA/Cas9, simultaneously. The DNA fragment demonstrated the presence of indels in target sites as well as provided the potential of CRISPR/Cas9 system. CONCLUSION: CRISPR/Cas9 offers a great potential as an efficient technique for editing of ZEB1 and ZEB2 genes in A549 cell line (Tab. 1, Fig. 6, Ref. 44).
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Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel.
BACKGROUND: CRISPR/Cas9 is an invaluable tool for studying cell biology and the development of molecular therapies. However, delivery of CRISPR/Cas9 components into some cell types remains a major hurdle. Primary human myoblasts are a valuable cell model for muscle studies, but are notoriously difficult to transfect. There are currently no commercial lipofection protocols tailored for primary myoblasts, and most generic guidelines simply recommend transfecting healthy cells at high confluency. This study aimed to maximize CRISPR/Cas9 transfection and editing in primary human myoblasts. METHODS: Since increased cell proliferation is associated with increased transfection efficiency, we investigated two factors known to influence myoblast proliferation: cell confluency, and a basement membrane matrix, Matrigel. CRISPR/Cas9 editing was performed by delivering Cas9 ribonucleoprotein complexes via lipofection into primary human myoblasts, cultured in wells with or without a Matrigel coating, at low (~ 40%) or high (~ 80%) confluency. RESULTS: Cells transfected at low confluency on Matrigel-coated wells had the highest levels of transfection, and were most effectively edited across three different target loci, achieving a maximum editing efficiency of 93.8%. On average, editing under these conditions was >4-fold higher compared to commercial recommendations (high confluency, uncoated wells). CONCLUSION: This study presents a simple, effective and economical method of maximizing CRISPR/Cas9-mediated gene editing in primary human myoblasts. This protocol could be a valuable tool for improving the genetic manipulation of cultured human skeletal muscle cells, and potentially be adapted for use in other cell types.
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High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris.
Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.
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An update on CRISPR-Cas12 as a versatile tool in genome editing.
Gene editing techniques, which help in modification of any DNA sequence at ease, have revolutionized the world of Genetic engineering. Although there are other gene-editing techniques, CRISPR has emerged as the chief and most preferred tool due to its simplicity and capacity to execute effective gene editing in a wide range of organisms. Although Cas9 has widely been employed for genetic modification over the years, Cas12 systems have lately emerged as a viable option. This review primarily focuses on assessing Cas12-mediated mutagenesis and elucidating the editing efficacy of both Cpf1 (Cas12a) and C2c1 (Cas12b) systems in microbes, plants, and other species. Also, we reviewed several genetic alterations that have been performed with these Cas12 systems to improve editing efficiency. Furthermore, the experimental benefits and applications of Cas12 systems are highlighted in this study.
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Targeted Gene Editing of Nuclear-Encoded Plastid Proteins in Phaeodactylum tricornutum via CRISPR/Cas9.
Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields.
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