Prime Editing for the Installation and Correction of Mutations Causing Inherited Retinal Disease: A Brief Methodology.

Inherited retinal diseases (IRDs) encompass a large heterogeneous group of rare blinding disorders whose etiology originates from mutations in the 280 genes identified to date. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems represent a promising avenue for the treatment of IRDs, as exemplified by FDA clinical trial approval of EDIT-101 (AGN-151587), which removes a deep intronic variant in the CEP290 gene that causes Leber congenital amaurosis (LCA) type 10. Prime editing is a novel double-strand break (DSB) independent CRISPR/Cas system which has the potential to correct all 12 possible transition and transversion mutations in addition to small deletions and insertions. Here, as a proof-of-concept study, we describe a methodology using prime editing for the in vitro installation and correction of the classical Pde6brd10 c.1678C > T (p.Arg560Cys) mutation which causes autosomal recessive retinitis pigmentosa (RP) in mice.

CRISPR/Cas9-based Gene Therapies for Fighting Drug Resistance Mediated by Cancer Stem Cells.

Cancer stem cells (CSCs) are cancer-initiating cells found in most tumors and hematological cancers. CSCs are involved in cells progression, recurrence of tumors, and drug resistance. Current therapies have been focused on treating the mass of tumor cells and cannot eradicate the CSCs. CSCs drug-specific targeting is considered as an approach to precisely target these cells. Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing systems are making progress and showing promise in the cancer research field. One of the attractive applications of CRISPR/Cas9 as one approach of gene therapy is targeting the critical genes involved in drug resistance and maintenance of CSCs. The synergistic effects of gene editing as a novel gene therapy approach and traditional therapeutic methods, including chemotherapy, can resolve drug resistance challenges and regression of the cancers. This review article considers different aspects of CRISPR/Cas9 ability in the study and targeting of CSCs with the intention to investigate their application in drug resistance.

RUNX2 Regulates Osteoblast Differentiation via the BMP4 Signaling Pathway.

RUNX2 is a master osteogenic transcription factor, and mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Studies have revealed that RUNX2 is not only a downstream target of the bone morphogenetic protein (BMP) pathway but can also regulate the expression of BMPs. However, the underlying mechanism of the regulation of BMPs by RUNX2 remains unknown. In this project, we diagnosed a CCD patient with a 7.86-Mb heterozygous deletion on chromosome 6 containing all exons of RUNX2 by multiplex ligation-dependent probe amplification (MLPA) and whole-genome sequencing (WGS). Bone marrow mesenchymal stem cells (BMSCs) were further extracted from patient alveolar bone fragments (CCD-BMSCs), an excellent natural model to explore the possible mechanism. The osteogenic differentiation ability of CCD-BMSCs was severely affected by RUNX2 heterozygous deletion. Also, BMP4 decreased most in BMP ligands, and CHRDL1, a BMP antagonist, was abnormally elevated in CCD-BMSCs. Furthermore, BMP4 treatment essentially rescued the osteogenic capacity of CCD-BMSCs, and RUNX2 overexpression reversed the abnormal expression of BMP4 and CHRDL1. Notably, we constructed CRISPR/Cas9 Runx2+/m MC3T3-E1 cells, which simulated a variant in CCD-BMSCs, to exclude the interference of other gene deletions and the heterogeneity of the genetic background of primary cells, and verified all findings from the CCD-BMSCs. Moreover, the luciferase reporter experiment showed that RUNX2 could inhibit the transcription of CHRDL1. Through immunofluorescence, the inhibitory effect of CHRDL1 on BMP4/Smad signaling was confirmed in MC3T3-E1 cells. These results revealed that RUNX2 regulated the BMP4 pathway by inhibiting CHRDL1 transcription. We collectively identified a novel RUNX2/CHRDL1/BMP4 axis to regulate osteogenic differentiation and noted that BMP4 might be a valuable therapeutic option for treating bone diseases.

The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets.

Human trypanosomiasis and leishmaniasis are vector-borne neglected tropical diseases caused by infection with the protozoan parasites Trypanosoma spp. and Leishmania spp., respectively. Once restricted to endemic areas, these diseases are now distributed worldwide due to human migration, climate change, and anthropogenic disturbance, causing significant health and economic burden globally. The current chemotherapy used to treat these diseases has limited efficacy, and drug resistance is spreading. Hence, new drugs are urgently needed. Phenotypic compound screenings have prevailed as the leading method to discover new drug candidates against these diseases. However, the publication of the complete genome sequences of multiple strains, advances in the application of CRISPR/Cas9 technology, and in vivo bioluminescence-based imaging have set the stage for advancing target-based drug discovery. This review analyses the limitations of the narrow pool of available drugs presently used for treating these diseases. It describes the current drug-based clinical trials highlighting the most promising leads. Furthermore, the review presents a focused discussion on the most important biological and pharmacological challenges that target-based drug discovery programs must overcome to advance drug candidates. Finally, it examines the advantages and limitations of modern research tools designed to identify and validate essential genes as drug targets, including genomic editing applications and in vivo imaging.

Prime Editing for Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation-inherited or de novo-with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.

Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives.

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector's expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.

Citric acid from Aspergillus niger: a comprehensive overview.

Microbial citric acid has high economic importance and widely used in beverage, food, detergents, cosmetics and pharmaceutical industries. The filamentous fungus Aspergillus niger is a work horse and important cell factory in industry for the production of citric acid. Although in-depth literatures and reviews have been published to explain the biochemistry, biotechnology and genetic engineering study of citric acid production by Aspergillus niger separately but the present review compiled, all the aspects with upto date brief summary of the subject describing microorganisms, substrates and their pre-treatment, screening, fermentation techniques, metabolic engineering, biochemistry, product recovery and numerous biotechnological application of citric acid for simple understanding of microbial citric acid production. The availability of genome sequence of this organism has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism. An attempt has been also made to address the molecular mechanisms and application of recent advanced techniques such as CRISPR/Cas9 systems in enhancement of citric acid production.

Reprint of: Deciphering and simulating models of radiation genotoxicity with CRISPR/Cas9 systems.

This short review explores the utility and applications of CRISPR/Cas9 systems in radiobiology. Specifically, in the context of experimentally simulating genotoxic effects of Ionizing Radiation (IR) to determine the contributions from DNA targets and 'Complex Double-Stranded Breaks' (complex DSBs) to the IR response. To elucidate this objective, this review considers applications of CRISPR/Cas9 on nuclear DNA targets to recognize the respective 'nucleocentric' response. The article also highlights contributions from mitochondrial DNA (mtDNA) - an often under-recognized target in radiobiology. This objective requires accurate experimental simulation of IR-like effects and parameters with the CRISPR/Cas9 systems. Therefore, the role of anti-CRISPR proteins in modulating enzyme activity to simulate dose rate - an important factor in radiobiology experiments is an important topic of this review. The applications of auxiliary domains on the Cas9 nuclease to simulate oxidative base damage and multiple stressor experiments are also topics of discussion. Ultimately, incorporation of CRISPR/Cas9 experiments into computational parameters in radiobiology models of IR damage and shortcomings to the technology are discussed as well. Altogether, the simulation of IR parameters and lack of damage to non-DNA targets in the CRISPR/Cas9 system lends this rapidly emerging tool as an effective model of IR induced DNA damage. Therefore, this literature review ultimately considers the relevance of complex DSBs to radiobiology with respect to using the CRISPR/Cas9 system as an effective experimental tool in models of IR induced effects.

Deciphering and simulating models of radiation genotoxicity with CRISPR/Cas9 systems.

This short review explores the utility and applications of CRISPR/Cas9 systems in radiobiology. Specifically, in the context of experimentally simulating genotoxic effects of Ionizing Radiation (IR) to determine the contributions from DNA targets and 'Complex Double-Stranded Breaks' (complex DSBs) to the IR response. To elucidate this objective, this review considers applications of CRISPR/Cas9 on nuclear DNA targets to recognize the respective 'nucleocentric' response. The article also highlights contributions from mitochondrial DNA (mtDNA) - an often under-recognized target in radiobiology. This objective requires accurate experimental simulation of IR-like effects and parameters with the CRISPR/Cas9 systems. Therefore, the role of anti-CRISPR proteins in modulating enzyme activity to simulate dose rate - an important factor in radiobiology experiments is an important topic of this review. The applications of auxiliary domains on the Cas9 nuclease to simulate oxidative base damage and multiple stressor experiments are also topics of discussion. Ultimately, incorporation of CRISPR/Cas9 experiments into computational parameters in radiobiology models of IR damage and shortcomings to the technology are discussed as well. Altogether, the simulation of IR parameters and lack of damage to non-DNA targets in the CRISPR/Cas9 system lends this rapidly emerging tool as an effective model of IR induced DNA damage. Therefore, this literature review ultimately considers the relevance of complex DSBs to radiobiology with respect to using the CRISPR/Cas9 system as an effective experimental tool in models of IR induced effects.

Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.

A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.

Cardiac tissue engineering: state-of-the-art methods and outlook.

The purpose of this review is to assess the state-of-the-art fabrication methods, advances in genome editing, and the use of machine learning to shape the prospective growth in cardiac tissue engineering. Those interdisciplinary emerging innovations would move forward basic research in this field and their clinical applications. The long-entrenched challenges in this field could be addressed by novel 3-dimensional (3D) scaffold substrates for cardiomyocyte (CM) growth and maturation. Stem cell-based therapy through genome editing techniques can repair gene mutation, control better maturation of CMs or even reveal its molecular clock. Finally, machine learning and precision control for improvements of the construct fabrication process and optimization in tissue-specific clonal selections with an outlook of cardiac tissue engineering are also presented.

Application of the CRISPR/Cas9 gene editing technique to research on functional genomes of parasites.

The clustered regularly-interspaced short palindromic repeats (CRISPR) structural family functions as an acquired immune system in prokaryotes. Gene editing techniques have co-opted CRISPR and the associated Cas nucleases to allow for the precise genetic modification of human cells, zebrafish, mice, and other eukaryotes. Indeed, this approach has been used to induce a variety of modifications including directed insertion/deletion (InDel) of bases, gene knock-in, introduction of mutations in both alleles of a target gene, and deletion of small DNA fragments. Thus, CRISPR technology offers a precise molecular tool for directed genome modification with a range of potential applications; further, its high mutation efficiency, simple process, and low cost provide additional advantages over prior editing techniques. This paper will provide an overview of the basic structure and function of the CRISPR gene editing system as well as current and potential applications to research on parasites.