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Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment.
In recent years, gene editing, especially that using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, has made great progress in the field of gene function. Rapid development of gene editing techniques has contributed to their significance in the field of medicine. Because the CRISPR/Cas9 gene editing tool is not only powerful but also has features such as strong specificity and high efficiency, it can accurately and rapidly screen the whole genome, facilitating the administration of gene therapy for specific diseases. In the field of tumor research, CRISPR/Cas9 can be used to edit genomes to explore the mechanisms of tumor occurrence, development, and metastasis. In these years, this system has been increasingly applied in tumor treatment research. CRISPR/Cas9 can be used to treat tumors by repairing mutations or knocking out specific genes. To date, numerous preliminary studies have been conducted on tumor treatment in related fields. CRISPR/Cas9 holds great promise for gene-level tumor treatment. Personalized and targeted therapy based on CRISPR/Cas9 will possibly shape the development of tumor therapy in the future. In this study, we review the findings of CRISPR/Cas9 for tumor treatment research to provide references for related future studies on the pathogenesis and clinical treatment of tumors.
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Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.
Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.
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The CRISPR/Cas9 Genome Editing Revolution.
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An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.
We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.
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Creating Heritable Mutations in Drosophila with CRISPR-Cas9.
Reverse genetics-the creation of mutations in preselected target genes-has until recently been a bottleneck in many Drosophila projects. The advent of clustered, regularly interspaced, short palindromic repeat (CRISPR) genome engineering systems has transformed this situation. A short time after the in vitro demonstration of target site cleavage by the RNA-guided endonuclease CRISPR-associated nuclease 9 (Cas9) (Jinek et al., Science 337:816-821, 2012), hundreds of fly researchers are using CRISPR technology to generate loss-of-function mutant alleles in specific genes, as well as to create specific point mutations or tagged protein products. It appears that most target genes can be edited with remarkably high efficiency, with engineered strains often available a few weeks after conception of a project. Here, we provide a step-by-step protocol for creating loss-of-function mutations in Drosophila using transgenic Cas9 sources, which is based on optimized reagents and procedures that have been evaluated in our laboratory. We also provide guidance on extending this protocol to produce precise genomic alterations by homology-directed repair in the presence of a donor sequence. Additional information and updates are available from our website, www.crisprflydesign.org .
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Global Governance of Human Genome Editing: What Are the Rules?
Human gene editing, particularly using the new CRISPR/Cas9 technology, will greatly increase the capability to make precise changes to human genomes. Human gene editing can be broken into four major categories: somatic therapy, heritable gene editing, genetic enhancement, and basic and applied research. Somatic therapy is generally well governed by national regulatory systems, so the need for global governance is less urgent. All nations are in agreement that heritable gene editing should not proceed at this time, but there is likely to be divergence if and when such procedures are shown to be safe and effective. Gene editing for enhancement purposes is not feasible today but is more controversial with the public, and many nations do not have well-developed regulatory systems for addressing genetic enhancement. Finally, different nations treat research with human embryos very differently based on deeply embedded social, cultural, ethical, and legal traditions. Several international governance mechanisms are currently in operation for human gene editing, and several other governance mechanisms have been proposed. It is unlikely that any single mechanism will alone be effective for governing human gene editing; rather, a polycentric or ecosystem approach that includes several overlapping and interacting components is likely to be necessary.
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Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.
Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.
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CRISPR-Cas gene editing technology and its application prospect in medicinal plants.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing technology has opened a new era of genome interrogation and genome engineering because of its ease operation and high efficiency. An increasing number of plant species have been subjected to site-directed gene editing through this technology. However, the application of CRISPR-Cas technology to medicinal plants is still in the early stages. Here, we review the research history, structural characteristics, working mechanism and the latest derivatives of CRISPR-Cas technology, and discussed their application in medicinal plants for the first time. Furthermore, we creatively put forward the development direction of CRISPR technology applied to medicinal plant gene editing. The aim is to provide a reference for the application of this technology to genome functional studies, synthetic biology, genetic improvement, and germplasm innovation of medicinal plants. CRISPR-Cas is expected to revolutionize medicinal plant biotechnology in the near future.
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One Small Dose, One Giant Leap for CRISPR Gene Editing.
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[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].
Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.
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