Genome engineering with targetable nucleases.

Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for their use in different biological systems.

Type III CRISPR-Cas: beyond the Cas10 effector complex.

Type III CRISPR-Cas loci encode some of the most abundant, yet complex, immune systems of prokaryotes. They are composed of a Cas10 complex that uses an RNA guide to recognize transcripts from bacteriophage and plasmid invaders. Target recognition triggers three activities within this complex: ssDNA degradation, synthesis of cyclic oligoadenylates (cOA) that act as second messengers to activate CARF-domain effectors, and cleavage of target RNA. This review covers recent research in type III CRISPR-Cas systems that looked beyond the activity of the canonical Cas10 complexes towards: (i) ancillary nucleases and understanding how they provide defense by sensing cOA molecules; (ii) ring nucleases and their role in regulating cOA production; and (iii) CRISPR-associated proteases, including the function of the Craspase complex in a transcriptional response to phage infection.

Nucleases Acting at Stalled Forks: How to Reboot the Replication Program with a Few Shortcuts.

In a lifetime, a human being synthesizes approximately 2×1016 meters of DNA, a distance that corresponds to 130,000 times the distance between the Earth and the Sun. This daunting task is executed by thousands of replication forks, which progress along the chromosomes and frequently stall when they encounter DNA lesions, unusual DNA structures, RNA polymerases, or tightly-bound protein complexes. To complete DNA synthesis before the onset of mitosis, eukaryotic cells have evolved complex mechanisms to process and restart arrested forks through the coordinated action of multiple nucleases, topoisomerases, and helicases. In this review, we discuss recent advances in understanding the role and regulation of nucleases acting at stalled forks with a focus on the nucleolytic degradation of nascent DNA, a process commonly referred to as fork resection. We also discuss the effects of deregulated fork resection on genomic instability and on the unscheduled activation of the interferon response under replication stress conditions.

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges.

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death.

A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1.

Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3'-5' exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics.

Development of mitochondrial gene-editing strategies and their potential applications in mitochondrial hereditary diseases: a review.

Mitochondrial DNA (mtDNA) is a critical genome contained within the mitochondria of eukaryotic cells, with many copies present in each mitochondrion. Mutations in mtDNA often are inherited and can lead to severe health problems, including various inherited diseases and premature aging. The lack of efficient repair mechanisms and the susceptibility of mtDNA to damage exacerbate the threat to human health. Heteroplasmy, the presence of different mtDNA genotypes within a single cell, increases the complexity of these diseases and requires an effective editing method for correction. Recently, gene-editing techniques, including programmable nucleases such as restriction endonuclease, zinc finger nuclease, transcription activator-like effector nuclease, clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated 9 and base editors, have provided new tools for editing mtDNA in mammalian cells. Base editors are particularly promising because of their high efficiency and precision in correcting mtDNA mutations. In this review, we discuss the application of these techniques in mitochondrial gene editing and their limitations. We also explore the potential of base editors for mtDNA modification and discuss the opportunities and challenges associated with their application in mitochondrial gene editing. In conclusion, this review highlights the advancements, limitations and opportunities in current mitochondrial gene-editing technologies and approaches. Our insights aim to stimulate the development of new editing strategies that can ultimately alleviate the adverse effects of mitochondrial hereditary diseases.

Gene Editing with Artificial DNA Scissors.

Artificial metallo-nucleases (AMNs) are small molecule DNA cleavage agents, also known as DNA molecular scissors, and represent an important class of chemotherapeutic with high clinical potential. This review provides a primary level of exploration on the concepts key to this area including an introduction to DNA structure, function, recognition, along with damage and repair mechanisms. Building on this foundation, we describe hybrid molecules where AMNs are covalently attached to directing groups that provide molecular scissors with enhanced or sequence specific DNA damaging capabilities. As this research field continues to evolve, understanding the applications of AMNs along with synthetic conjugation strategies can provide the basis for future innovations, particularly for designing new artificial gene editing systems.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Recent Advances in Tomato Gene Editing.

The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification of physiological, morphological, and other characteristics in a wide range of crops to mitigate the negative effects of stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have the potential to improve crop resilience and increase yields in response to challenging environmental conditions. This review provides an overview of gene-editing techniques used in plants, focusing on the cultivated tomatoes. Several dozen genes that have been successfully edited with the CRISPR/Cas system were selected for inclusion to illustrate the possibilities of this technology in improving fruit yield and quality, tolerance to pathogens, or responses to drought and soil salinity, among other factors. Examples are also given of how the domestication of wild species can be accelerated using CRISPR/Cas to generate new crops that are better adapted to the new climatic situation or suited to use in indoor agriculture.

Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples.

The impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing offers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modification of plant's genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the final product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01423-y.

Stability of DNA Origami Nanostructures in Physiological Media: The Role of Molecular Interactions.

Programmable, custom-shaped, and nanometer-precise DNA origami nanostructures have rapidly emerged as prospective and versatile tools in bionanotechnology and biomedicine. Despite tremendous progress in their utilization in these fields, essential questions related to their structural stability under physiological conditions remain unanswered. Here, DNA origami stability is explored by strictly focusing on distinct molecular-level interactions. In this regard, the fundamental stabilizing and destabilizing ionic interactions as well as interactions involving various enzymes and other proteins are discussed, and their role in maintaining, modulating, or decreasing the structural integrity and colloidal stability of DNA origami nanostructures is summarized. Additionally, specific issues demanding further investigation are identified. This review - through its specific viewpoint - may serve as a primer for designing new, stable DNA objects and for adapting their use in applications dealing with physiological media.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia.

Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from Verrucomicrobia bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.

Cellular Responses to Widespread DNA Replication Stress.

Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants.

Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2 , respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1 , we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice.

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

Ultra-specific nucleic acid testing by target-activated nucleases.

Specific and sensitive detection of nucleic acids is essential to clinical diagnostics and biotechnological applications. Currently, amplification steps are necessary for most detection methods due to the low concentration of nucleic acid targets in real samples. Although amplification renders high sensitivity, poor specificity is prevalent because of the lack of highly accurate precise strategies, resulting in significant false positives and false negatives. Nucleases exhibit high catalytic activity for nucleic acid cleavage which is regulated in a programmable manner. This review focuses on the latest progress in nucleic acid testing methods based on the target-activated nucleases. It summarizes the property of enzymes such as CRISPR/Cas, Argonautes, and some gene-editing irrelevant nucleases, which have been leveraged to create highly specific and sensitive nucleic acid testing tools. We elaborate on recent advances in the field of nuclease-mediated DNA recognition techniques for nucleic acid detection, and discuss its future applications and challenges in molecular diagnostics.

Elucidation of CRISPR-Cas9 application in novel cellular immunotherapy.

Novel cellular immunotherapy with engineered T cells has improved cancer treatment and established therapeutic promises to prevent tumor formation in clinical studies. Due to certain restrictions and difficulties, CAR and TCR T-cells therapies were inadequate at points. CRISPR Cas9 genome-editing tool has significant potential for these two cell-based therapies. As a specialized gene-editing technique, CRISPR Cas9 is used to repair genetic alternations with minimal damage. It is used as an adjunct to immunotherapy to stimulate a more robust immune response. CRISPR has long outpaced other target-specific genome editing methods such as ZFNs and TALEN because of its high efficiency, competence in targeting, and stable operating conditions. CRISPR can overcome the two major drawbacks of universal CAR T cells: allorejection and graft-vs-host disease. TCR-based T cell treatment can reduce inappropriate binding between endogenous and transgenic TCR, resulting in a reduction of severe toxicity. The CAR and TCR T based cell therapies uphold an excellent future for tumor malignancies. This article has elucidated the administration of CRISPR Cas9 in novel cellular immunotherapy, CAR, and TCR T cell therapy. However, this article did not fail to observe this technology's ethical concerns, limitations, and challenges. Furthermore, the article compares CRISPR-mediated allogeneic CAR T cell to TCR-T cell therapy.

The Role of Nucleases Cleaving TLR3, TLR7/8 and TLR9 Ligands, Dicer RNase and miRNA/piRNA Proteins in Functional Adaptation to the Immune Escape and Xenophagy of Prostate Cancer Tissue.

The prototypic sensors for the induction of innate and adaptive immune responses are the Toll-like receptors (TLRs). Unusually high expression of TLRs in prostate carcinoma (PC), associated with less differentiated, more aggressive and more propagating forms of PC, changed the previous paradigm about the role of TLRs strictly in immune defense system. Our data reveal an entirely novel role of nucleic acids-sensing Toll-like receptors (NA-TLRs) in functional adaptation of malignant cells for supply and digestion of surrounding metabolic substrates from dead cells as specific mechanism of cancer cells survival, by corresponding ligands accelerated degradation and purine/pyrimidine salvage pathway. The spectrophotometric measurement protocols used for the determination of the activity of RNases and DNase II have been optimized in our laboratory as well as the enzyme-linked immunosorbent method for the determination of NF-κB p65 in prostate tissue samples. The protocols used to determine Dicer RNase, AGO2, TARBP2 and PIWIL4 were based on enzyme-linked immunosorbent assay. The amount of pre-existing acid-soluble oligonucleotides was measured and expressed as coefficient of absorbance. The activities of acid DNase II and RNase T2, and the activities of nucleases cleaving TLR3, TLR7/8 and TLR9 ligands (Poly I:C, poly U and unmethylated CpG), increased several times in PC, compared to the corresponding tumor adjacent and control tissue, exerting very high sensitivity and specificity of above 90%. Consequently higher levels of hypoxanthine and NF-κB p65 were reported in PC, whereas the opposite results were observed for miRNA biogenesis enzyme (Dicer RNase), miRNA processing protein (TARB2), miRNA-induced silencing complex protein (Argonaute-AGO) and PIWI-interacting RNAs silence transposon. Considering the crucial role of purine and pyrimidine nucleotides as energy carriers, subunits of nucleic acids and nucleotide cofactors, future explorations will be aimed to design novel anti-cancer immune strategies based on a specific acid endolysosomal nuclease inhibition.