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Current progress in CRISPR-Cas systems for cancer.
Cancer has been a primary contributor to morbidity and mortality worldwide. With an increasing trend of incidence and prevalence of cancer, progress has also been made in its treatment, starting from radiation and chemotherapy to immunotherapy and gene therapy. CRISPR-Cas technique, a promising gene editing tool, has been employed in cancer research for novel treatment regimens, identification of therapeutic targets, and unraveling the genetic mechanisms behind oncogenesis. CRISPR-based genome editing helped in identifying the roles of specific genetic factors linked to treatment resistance, metastasis, and cancer development. CRISPR allows the discovery of genes and treatment options through specifically interrupting tumor activators or activating tumor suppressor genes in cancer cells. Advancements in CRISPR technology, especially the use of immune cells like chimeric antigen receptor (CAR) T cells, has the potential to revolutionize personalized cancer treatment by precisely targeting and killing cancer cells. Furthermore, reactivating tumor suppressor genes makes cancer cells more susceptible to chemotherapy or immunotherapy. CRISPR-mediated genome editing can, hence, help to overcome resistance to traditional cancer treatments. The current manuscript covers that how is the CRISPR technology propelling revolutionary development in the field of cancer research, providing advance perspectives on the molecular causes of the disease and creating new lines for the development of more precise and potent cancer therapies.
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Genome Editing for Developing Disease-Resistant Plants.
Genome editing tools, most notably the CRISPR-Cas9 system, have transformed modern crop breeding by allowing precise and efficient modification of plant genomes, particularly for enhancing resistance to disease. In this chapter, we present two major CRISPR-based approaches that have been successfully applied to develop disease-resistant crops: (1) gene knockout of host susceptibility (S) genes to block pathogen compatibility, and (2) targeted insertion of resistance alleles or synthetic genes using the homology-directed repair (HDR) pathway. Both strategies utilize the specificity and efficiency of CRISPR-Cas9 to introduce defined edits in crops such as rice. This chapter outlines comprehensive step-by-step protocols covering key elements of the process, including target site selection, construction of sgRNA and Cas9 vectors, plant transformation methods, and mutation validation techniques. Together, these methods provide a powerful platform for engineering plant immune responses and offer practical tools to accelerate the breeding of disease-resistant cultivars.
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Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.
Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.
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Genome Editing Dilemma: Navigating Dual-Use Potential and Charting the Path Forward.
Contemporary genome editing techniques have made genomic intervention-from microorganism to human-more accessible, easier to use, and more accurate than previous methods. We argue that, notwithstanding its merits in treating and preventing disease in humans, genome editing represents a potential threat for domestic and international security, requiring an integrated approach in regulating, detecting, preventing, and mitigating the risk of its use for malicious purposes. Despite the global regulatory ambitions of the 2021 WHO framework, we see insufficient attention given to the future prospect of dual-use genomic technology. Drawing parallels with the nuclear field, we suggest tentative practical steps for a way forward in dealing with genome editing technologies, such as: 1) adapting national (bio)security and defence strategies to include genome editing as a possible threat (with conceivable WMD potential); 2) enhancing the international dialogue on genome editing and raising the issue at the highest level; 3) working towards a global, legally binding verification mechanism; 4) tracking genome editing technologies.
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[CRISPR-Cas9 editing of HIV-1 neutralizing human B cells].
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An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells.
Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.
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Strategies and Protocols for Optimized Genome Editing in Potato.
The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.
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Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9.
Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.
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Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration.
Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6 bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.
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CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing.
The development of a method for genome editing based on CRISPR-Cas9 technology was awarded The Nobel Prize in Chemistry in 2020, less than a decade after the discovery of all principal molecular components of the system. For the first time in history a Nobel prize was awarded to two women, Emmanuelle Charpentier and Jennifer Doudna, who made key discoveries in the field of DNA manipulation with the CRISPR-Cas9 system, so-called "genetic scissors". It is difficult to overestimate the importance of the technique as it enables one not only to manipulate genomes of model organisms in scientific experiments, and modify characteristics of important crops and animals, but also has the potential of introducing revolutionary changes in medicine, especially in treatment of genetic diseases. The original biological function of CRISPR-Cas9 system is the protection of prokaryotes from mobile genetic elements, in particular viruses. Currently, CRISPR-Cas9 and related technologies have been successfully used to cure life-threatening diseases, make coronavirus detection tests, and even to modify human embryo cells with the consequent birth of babies carrying the introduced modifications. This intervention with human germplasm cells resulted in wide disapproval in the scientific community due to ethical concerns, and calls for a moratorium on inheritable genomic manipulations. This review focuses on the history of the discovery of the CRISPR-Cas9 system with some aspects of its current applications, including ethical concerns about its use in humans.
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