Tool For Precise Genome Editing Research Articles

O-002 CRISPR-Cas, from a bacterial defense system to a novel genetic tool in humans

Abstract In a span of merely a decade, the CRISPR-Cas technology has revolutionized the scientific, industrial, and medical sectors. Its ability to serve as a highly precise genome editing tool has gained a lot of attention over these years, leading to its recognition with the Nobel Prize in Chemistry in 2020. Amidst these achievements, it is easy to overlook the natural role of the CRISPR-Cas system, which functions as an adaptive immune system against viruses in bacteria. The ongoing exploration and characterization of various variants of these systems continue to pave the way for the development of innovative genetic tools and the enhancement of existing ones. In this presentation, I will start with an explanation of the natural role of CRISPR-Cas in bacteria. This will be followed by a brief overview of how these systems were repurposed into the aforementioned genome editing tools. Subsequently, I will delve into some of the latest advancements (and their bottlenecks) in the field, including a few unconventional applications, and discuss their potential short-term and long-term impacts on human reproduction.

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.

Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.

Scaffold-based non-viral CRISPR delivery platform for efficient and prolonged gene activation to accelerate tissue regeneration

Clustered regularly interspaced short palindromic repeat activation (CRISPRa) technology has emerged as a precise genome editing tool for activating endogenous transgene expression. While it holds promise for precise cell modification, its translation into tissue engineering has been hampered by biosafety concerns and suboptimal delivery methods. To address these challenges, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. The Gel/NF scaffold facilitates the controlled and sustained release of CRISPRa complexes and also promotes cell recruitment to the scaffold for efficient and localized transfection. As a proof of concept, we employed this CRISPRa delivery platform to activate the vascular endothelial growth factor (VEGF) gene in a rat model with full-thickness skin defects. Our results demonstrate sustained upregulation of VEGF expression even at 21 days post-implantation, resulting in enhanced angiogenesis and improved skin regeneration. These findings underscore the potential of the Gel/NF scaffold-based CRISPRa delivery platform as an efficient and durable strategy for gene activation, offering promising prospects for tissue regeneration. Statement of significanceTranslation of clustered regularly interspaced short palindromic repeat activation (CRISPRa) therapy to tissue engineering is limited by biosafety concerns and unsatisfactory delivery strategy. To solve this issue, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. This scaffold enables controlled and sustained release of CRISPRa and can induce cell recruitment for localized transfection. As a proof of concept, we activated vascular endothelial growth factor (VEGF) in a rat model with full-thickness skin defects, leading to sustained upregulation of VEGF expression, enhanced angiogenesis and improved skin regeneration in vivo. These findings demonstrate the potential of this platform for gene activation, thereby offering promising prospects for tissue regeneration.

CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.).

Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.). In the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection. Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.

Deep learning in CRISPR-Cas systems: a review of recent studies.

In genetic engineering, the revolutionary CRISPR-Cas system has proven to be a vital tool for precise genome editing. Simultaneously, the emergence and rapid evolution of deep learning methodologies has provided an impetus to the scientific exploration of genomic data. These concurrent advancements mandate regular investigation of the state-of-the-art, particularly given the pace of recent developments. This review focuses on the significant progress achieved during 2019-2023 in the utilization of deep learning for predicting guide RNA (gRNA) activity in the CRISPR-Cas system, a key element determining the effectiveness and specificity of genome editing procedures. In this paper, an analytical overview of contemporary research is provided, with emphasis placed on the amalgamation of artificial intelligence and genetic engineering. The importance of our review is underscored by the necessity to comprehend the rapidly evolving deep learning methodologies and their potential impact on the effectiveness of the CRISPR-Cas system. By analyzing recent literature, this review highlights the achievements and emerging trends in the integration of deep learning with the CRISPR-Cas systems, thus contributing to the future direction of this essential interdisciplinary research area.

Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.

Precision Editing as a Therapeutic Approach for β-Hemoglobinopathies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

Editing Aspergillus terreus using the CRISPR-Cas9 system.

CRISPR-Cas9 technology has been utilized in different organisms for targeted mutagenesis, offering a fast, precise and cheap approach to speed up molecular breeding and study of gene function. Until now, many researchers have established the demonstration of applying the CRISPR/Cas9 system to various fungal model species. However, there are very few guidelines available for CRISPR/Cas9 genome editing in Aspergillus terreus. In this study, we present CRISPR/Cas9 genome editing in A. terreus. To optimize the guide ribonucleic acid (gRNA) expression, we constructed a modified single-guide ribonucleic acid (sgRNA)/Cas9 expression plasmid. By co-transforming an sgRNA/Cas9 expression plasmid along with maker-free donor deoxyribonucleic acid (DNA), we precisely disrupted the lovB and lovR genes, respectively, and created targeted gene insertion (lovF gene) and iterative gene editing in A. terreus (lovF and lovR genes). Furthermore, co-delivering two sgRNA/Cas9 expression plasmids resulted in precise gene deletion (with donor DNA) in the ku70 and pyrG genes, respectively, and efficient removal of the DNA between the two gRNA targeting sites (no donor DNA) in the pyrG gene. Our results showed that the CRISPR/Cas9 system is a powerful tool for precise genome editing in A. terreus, and our approach provides a great potential for manipulating targeted genes and contributions to gene functional study of A. terreus.

An Alternate Approach to Generate Induced Pluripotent Stem Cells with Precise CRISPR/Cas9 Tool.

The induced pluripotent stem cells (iPSCs) are considered powerful tools in pharmacology, biomedicine, toxicology, and cell therapy. Multiple approaches have been used to generate iPSCs with the expression of reprogramming factors. Here, we generated iPSCs by integrating the reprogramming cassette into a genomic safe harbor, CASH-1, with the use of a precise genome editing tool, CRISPR/Cas9. The integration of cassette at CASH-1 into target cells did not alter the pattern of proliferation and interleukin-6 secretion as a response to ligands of multiple signaling pathways involving tumor necrosis factor-α receptor, interleukin-1 receptor, and toll-like receptors. Moreover, doxycycline-inducible expression of OCT4, SOX2, and KLF4 reprogrammed engineered human dermal fibroblasts and human embryonic kidney cell line into iPSCs. The generated iPSCs showed their potential to make embryoid bodies and differentiate into the derivatives of all three germ layers. Collectively, our data emphasize the exploitation of CASH-1 by CRISPR/Cas9 tool for therapeutic and biotechnological applications including but not limited to reprogramming of engineered cells into iPSCs.

Engineering crop resistance by manipulating disease susceptibility genes

Wheat production is often affected by different types of rust diseases, in which stripe rust is triggered by a fungal pathogen, Puccinia striiformis f. sp. tritici (Pst), and remains the biggest challenge to the plant breeders and farmer community. Nevertheless, pathogens exploit its effector proteins for altering the susceptibility (S) genes in the host plant, rendering host defense inactivated. Thus, altering the S genes by employing precise genome editing tools provides a great opportunity to develop broad-spectrum and durable resistant crops. Recently, Wang et. al. 2022 have demonstrated the TaPsIPK1 as an ideal S gene target to confer stripe rust resistance in wheat, providing an opportunity to deliver broad spectrum rust-resistant crop varieties.

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Wheat genomic study for genetic improvement of traits in China.

Bread wheat (Triticum aestivum L.) is a major crop that feeds 40% of the world's population. Over the past several decades, advances in genomics have led to tremendous achievements in understanding the origin and domestication of wheat, and the genetic basis of agronomically important traits, which promote the breeding of elite varieties. In this review, we focus on progress that has been made in genomic research and genetic improvement of traits such as grain yield, end-use traits, flowering regulation, nutrient use efficiency, and biotic and abiotic stress responses, and various breeding strategies that contributed mainly by Chinese scientists. Functional genomic research in wheat is entering a new era with the availability of multiple reference wheat genome assemblies and the development of cutting-edge technologies such as precise genome editing tools, high-throughput phenotyping platforms, sequencing-based cloning strategies, high-efficiency genetic transformation systems, and speed-breeding facilities. These insights will further extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process, ultimately contributing to more sustainable agriculture in China and throughout the world.

CRISPR/Cas9 is a powerful tool for precise genome editing of legume crops: a review.

Legumes are an imperative source of food and proteins across the globe. They also improve soil fertility through symbiotic nitrogen fixation (SNF). Genome editing (GE) is now a novel way of developing desirable traits in legume crops. Genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR) system permits a defined genome alteration to improve crop performance. This genome editing tool is reliable, cost-effective, and versatile, and it has to deepen in terms of use compared to other tools. Recently, many novel variations have drawn the attention of plant geneticists, and efforts are being made to develop trans-gene-free cultivars for ensuring biosafety measures. This review critically elaborates on the recent development in genome editing of major legumes crops. We hope this updated review will provide essential informations for the researchers working on legumes genome editing. In general, the CRISPR/Cas9 novel GE technique can be integrated with other techniques like omics approaches and next-generation tools to broaden the range of gene editing and develop any desired legumes traits. Regulatory ethics of CRISPR/Cas9 are also discussed.

CRISPR/Cas9-Mediated Targeted Mutagenesis of FtMYB45 Promotes Flavonoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum).

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is an efficient genome editing tool used in multiple plant species. However, it has not been applied to Tartary buckwheat (Fagopyrum tataricum), which is an important edible and medicinal crop rich in rutin and other flavonoids. FtMYB45 is an R2R3-type MYB transcription factor that negatively regulates flavonoid biosynthesis in Tartary buckwheat. Here, the CRISPR/Cas9 system polycistronic tRNA-sgRNA (PTG)/Cas9 was employed to knock out the FtMYB45 gene in Tartary buckwheat. Two single-guide RNAs (sgRNAs) were designed to target the second exon of the FtMYB45 gene. Twelve transgenic hairy roots were obtained using Agrobacterium rhizogenes-mediated transformation. Sequencing data revealed that six lines containing six types of mutations at the predicted double-stranded break site were generated using sgRNA1. The mutation frequency reached 50%. A liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ-MS) based metabolomic analysis revealed that the content of rutin, catechin, and other flavonoids was increased in hairy root mutants compared with that of lines transformed with the empty vector. Thus, CRISPR/Cas9-mediated targeted mutagenesis of FtMYB45 effectively increased the flavonoids content of Tartary buckwheat. This finding demonstrated that the CRISPR/Cas9 system is an efficient tool for precise genome editing in Tartary buckwheat and lays the foundation for gene function research and quality improvement in Tartary buckwheat.

Utilization of CRISPR-Mediated Tools for Studying Functional Genomics in Hematological Malignancies: An Overview on the Current Perspectives, Challenges, and Clinical Implications.

Hematological malignancies (HM) are a group of neoplastic diseases that are usually heterogenous in nature due to the complex underlying genetic aberrations in which collaborating mutations enable cells to evade checkpoints that normally safeguard it against DNA damage and other disruptions of healthy cell growth. Research regarding chromosomal structural rearrangements and alterations, gene mutations, and functionality are currently being carried out to understand the genomics of these abnormalities. It is also becoming more evident that cross talk between the functional changes in transcription and proteins gives the characteristics of the disease although specific mutations may induce unique phenotypes. Functional genomics is vital in this aspect as it measures the complete genetic change in cancerous cells and seeks to integrate the dynamic changes in these networks to elucidate various cancer phenotypes. The advent of CRISPR technology has indeed provided a superfluity of benefits to mankind, as this versatile technology enables DNA editing in the genome. The CRISPR-Cas9 system is a precise genome editing tool, and it has revolutionized methodologies in the field of hematology. Currently, there are various CRISPR systems that are used to perform robust site-specific gene editing to study HM. Furthermore, experimental approaches that are based on CRISPR technology have created promising tools for developing effective hematological therapeutics. Therefore, this review will focus on diverse applications of CRISPR-based gene-editing tools in HM and its potential future trajectory. Collectively, this review will demonstrate the key roles of different CRISPR systems that are being used in HM, and the literature will be a representation of a critical step toward further understanding the biology of HM and the development of potential therapeutic approaches.

Rice functional genomics: decades' efforts and roads ahead.

Rice (Oryza sativa L.) is one of the most important crops in the world. Since the completion of rice reference genome sequences, tremendous progress has been achieved in understanding the molecular mechanisms on various rice traits and dissecting the underlying regulatory networks. In this review, we summarize the research progress of rice biology over past decades, including omics, genome-wide association study, phytohormone action, nutrient use, biotic and abiotic responses, photoperiodic flowering, and reproductive development (fertility and sterility). For the roads ahead, cutting-edge technologies such as new genomics methods, high-throughput phenotyping platforms, precise genome-editing tools, environmental microbiome optimization, and synthetic methods will further extend our understanding of unsolved molecular biology questions in rice, and facilitate integrations of the knowledge for agricultural applications.

High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici.

Pediococcus acidilactici is commonly used for pediocin production and lactic acid fermentation. However, a high-efficiency genome editing tool is unavailable for this species. In this study, we constructed endogenous subtype II-A CRISPR-Cas system-based genome interference plasmids which carried a "repeat-spacer-repeat" cassette in the pMG36e shuttle vector. These plasmids exhibited self-interference activities in P. acidilactici LA412. Then, the genome-editing plasmids were constructed by cloning the upstream/downstream donor DNA into the corresponding interference plasmids to exert high-efficiency markerless gene deletion, gene integration, and point mutation in P. acidilactici LA412. We found that endogenous CRISPR-mediated depletion of the native plasmids enhanced the cell growth and that integration of an l-lactate dehydrogenase gene into the chromosome enhanced both cell growth and lactic acid production. IMPORTANCE A rapid and precise genome editing tool will promote the practical application of Pediococcus acidilactici, one type of lactic acid bacterium with excellent stress tolerance and probiotic characteristics. This study established a high-efficiency endogenous CRISPR-Cas system-based genome editing tool for P. acidilactici and achieved different genetic manipulations, including gene deletion, gene insertion, mononucleotide mutation, and endogenous plasmid depletion. The engineered strain edited by this tool showed significant advantages in cell growth and lactic acid fermentation. Therefore, our tool can satisfy the requirements for genetic manipulations of P. acidilactici, thus making it a sophisticated chassis species for synthetic biology and bioindustry.

Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science.

The recent discovery and subsequent development of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease.

Genome editing of avian species: implications for animal use and welfare.

Avian species are used as model systems in research and have contributed to ground-breaking concepts in developmental biology, immunology, genetics, virology, cancer and cell biology. The chicken in particular is an important research model and an agricultural animal as a major contributor to animal protein resources for the global population. The development of genome editing methods, including CRISPR/Cas9, to mediate germline engineering of the avian genome will have important applications in biomedical, agricultural and biotechnological activities. Notably, these precise genome editing tools have the potential to enhance avian health and productivity by identifying and validating beneficial genetic variants in bird populations. Here, we present a concise description of the existing methods and current applications of the genome editing tools in bird species, focused on chickens, with attention on animal use and welfare issues for each of the techniques presented.

Base editing in rice: current progress, advances, limitations, and future perspectives.

Base editing is one of the promising genome editing tools for generating single-nucleotide changes in rice genome. Rice (Oryza sativa L.) is an important staple food crop, feeding half of the population around the globe. Developing new rice varieties with desirable agronomic traits is necessary for sustaining global food security. The use of genome editing technologies for developing rice varieties is pre-requisite in the present scenario. Among the genome editing technologies developed for rice crop improvement, base editing technology has emerged as an efficient and reliable tool for precise genome editing in rice plants. Base editing technology utilizes either adenosine or cytidine base editor for precise editing at the target region. A base editor (adenosine or cytidine) is a fusion of catalytically inactive CRISPR/Cas9 domain and adenosine or cytidine deaminase domain. In this review, authors have discussed the different adenine and cytosine base editors developed so far for precise genome editing of rice via base editing technology. We address the current progress, advances, limitations, as well as future perspectives of the base editing technology for rice crop improvement.