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Developing a multi-modular assembled prime editing (mPE) system improved precise multi-base insertion efficiency in dicots.
INTRODUCTION: The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE efficiency severely restricts its application, especially in dicots. PE can introduce small tags to trace target protein or cis-element to regulate gene transcription which is an expertise superior to other gene editing tools. Owing to low efficiency, PE adaption in stably transformed Arabidopsis is lacking. OBJECTIVES: This study aimed to investigate the issue of low PE efficiency in dicots and develop systematic solutions to improve it. Currently, PE in dicots is undetectable and inconsistent, and this study seeks to address it. Split PE into several parts showed better performance in some target sites in mammal cells. We plan to discover the optimal split PE combination in dicot. METHODS: We conducted large-scale transformation experiments in dicot model plants Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by Agrobacterium-mediated transformation with deep amplicon sequencing (0.2-0.5 million clean total reads). RESULTS: The editing efficiency decreased upon using a fused reverse transcriptase (RT) or an extended pegRNA separately and further decreased dramatically when these were used together. With the help of the pol II strategy to express PE gRNA (pegRNA), we named the most effective split PE combination as a multi-modular assembled prime editing system (mPE). mPE exhibited improved precise editing efficiency on most gene sites with various editing types, ranging from 1.3-fold to 1288.5-fold and achieved PE on some sites that could not be edited by original PE2. Especially, mPE showed superiority for multi-base insertion with an average improvement of 197.9-fold. CONCLUSION: The original PE architecture strongly inhibited the cleavage activity of Cas9. Split PE improved PE efficiency extensively and was in favor of introducing small insertions in dicot plants, indicating that different PE variants might have their own expertise.
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Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.
The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.
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Therapeutic promise of CRISPR-Cas9 gene editing in sickle cell disease and β-thalassemia: A current review.
Sickle cell disease (SCD and β-thalassemia (BT) affects millions of people worldwide. In addition, around 500,000 infants are born with SCD and 60,000 people are diagnosed with BT every year. Mutations in the hemoglobin subunit beta (HBB) gene are responsible for causing both BT and SCD. Indeed, the diversity of potential mutations in the HBB gene elucidates the diversity in clinical severity observed in individuals with BT and related morbidities. On the other hand, SCD takes place because of the alteration in a single amino acid at position 6 in the beta-globin chain, where a base substitution occurs from glutamic acid to valine, which eventually results in abnormal sickle hemoglobin. Conventional therapies for BT and SCD including pharmaceutical drugs and blood transfusion might temporarily improve the clinical severity of these diseases, however these therapies cannot cure the diseases. CRISPR-Cas9 (CC9) is revolutionizing genome engineering, offering promising therapeutic avenues for genetic diseases. Therefore, CC9-mediated gene therapy provides great hope in the treatment of both BT and SCD. CC9-mediated gene therapy has already demonstrated its effectiveness in correcting both SCD and BT-causing mutations. Moreover, CC9-mediated gene editing was found to be effective in reactivating the expression of hemoglobin F (HbF) and regulating LRF and BCL11A. A number of clinical trials with CC9 gene-edited therapies are being carried out to elucidate their potential in treating BT and SCD. Genetics and pathophysiological mechanisms of SCD and BT, the mechanism of CC9-mediated gene editing, and common delivery methods of the CC9 system have been discussed in this review. Moreover, an in-depth discussion on applications and the current status of CC9-mediated gene editing in SCD and BT along with current challenges and future perspectives have been provided.
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Simultaneous knockout of multiple LHCF genes using single sgRNAs and engineering of a high-fidelity Cas9 for precise genome editing in marine algae.
The CRISPR/Cas9 system is an RNA-guided sequence-specific genome editing tool, which has been adopted for single or multiple gene editing in a wide range of organisms. When working with gene families with functional redundancy, knocking out multiple genes within the same family may be required to generate a phenotype. In this study, we tested the possibility of exploiting the known tolerance of Cas9 for mismatches between the single-guide RNA (sgRNA) and target site to simultaneously introduce indels in multiple homologous genes in the marine diatom Phaeodactylum tricornutum. As a proof of concept, we designed two sgRNAs that could potentially target the same six light-harvesting complex (LHC) genes belonging to the LHCF subgroup. Mutations in up to five genes were achieved simultaneously using a previously established CRISPR/Cas9 system for P. tricornutum. A visible colour change was observed in knockout mutants with multiple LHCF lesions. A combination of pigment, LHCF protein and growth analyses was used to further investigate the phenotypic differences between the multiple LHCF mutants and WT. Furthermore, we used the two same sgRNAs in combination with a variant of the existing Cas9 where four amino acids substitutions had been introduced that previously have been shown to increase Cas9 specificity. A significant reduction of off-target editing events was observed, indicating that the altered Cas9 functioned as a high-fidelity (HiFi) Cas9 nuclease.
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A primer to gene therapy: Progress, prospects, and problems.
Genetic therapies based on gene addition have witnessed a variety of clinical successes and the first therapeutic products have been approved for clinical use. Moreover, innovative gene editing techniques are starting to offer new opportunities in which the mutations that underlie genetic diseases can be directly corrected in afflicted somatic cells. The toolboxes underpinning these DNA modifying technologies are expanding with great pace. Concerning the ongoing efforts for their implementation, viral vector-based gene delivery systems have acquired center-stage, providing new hopes for patients with inherited and acquired disorders. Specifically, the application of genetic therapies using viral vectors for the treatment of inborn metabolic disorders is growing and clinical applications are starting to appear. While the field has matured from the technology perspective and has yielded efficacious products, it is the perception of many stakeholders that from the regulatory side further developments are urgently needed. In this review, we summarize the features of state-of-the-art viral vector systems and the corresponding gene-centered therapies they seek to deliver. Moreover, a brief summary is also given on emerging gene editing approaches built on CRISPR-Cas9 nucleases and, more recently, nickases, including base editors and prime editors. Finally, we will point at some regulatory aspects that may deserve further attention for translating these technological developments into actual advanced therapy medicinal products (ATMPs).
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A CRISPR/Cas12a-Mediated Sensitive DNA Detection System for Gene-Edited Rice.
BACKGROUND: In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing. OBJECTIVE: We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products. METHODS: In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods. RESULTS: The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products. CONCLUSION: We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice. HIGHLIGHTS: The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.
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Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses.
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.
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CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion.
BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.
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Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement.
The CRISPR-based gene editing technology has become more and more powerful in genome manipulation for agricultural breeding, with numerous improved toolsets springing up. In recent years, many CRISPR toolsets for gene editing, such as base editors (BEs), CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and plant epigenetic editors (PEEs), have been developed to clarify gene function and full-level gene regulation. Here, we comprehensively summarize the application and capacity of the different CRISPR toolsets in the study of plant gene expression regulation, highlighting their potential application in gene regulatory networks' analysis. The general problems in CRISPR application and the optimal solutions in the existing schemes for high-throughput gene function analysis are also discussed. The CRISPR toolsets targeting gene manipulation discussed here provide new solutions for further genetic improvement and molecular breeding of crops.
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Therapeutic Gene Editing in Inherited Retinal Disorders.
Since the development of CRISPR/Cas9 gene editing in 2012, therapeutic editing research has produced several phase 1-2a trials. Here we provide an overview of the mechanisms and applications of various gene-editing technologies including adeno-associated virus vectors, lentiviruses, CRISPR/Cas9 systems, base and prime editing, antisense oligonucleotides, short-hairpin RNAs, Cas13, and adenosine deaminase acting on RNA for the treatment of various inherited retinal diseases (IRDs). We outline the various stages of clinical trials using these technologies and the impacts they have made in advancing the practice of medicine.
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