The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Integrating Prime Editing and Cellular Reprogramming as Novel Strategies for Genetic Cardiac Disease Modeling and Treatment.

PURPOSE OF REVIEW: This review aims to evaluate the potential of CRISPR-based gene editing tools, particularly prime editors (PE), in treating genetic cardiac diseases. It seeks to answer how these tools can overcome current therapeutic limitations and explore the synergy between PE and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for personalized medicine. RECENT FINDINGS: Recent advancements in CRISPR technology, including CRISPR-Cas9, base editors, and PE, have demonstrated precise genome correction capabilities. Notably, PE has shown exceptional precision in correcting genetic mutations. Combining PE with iPSC-CMs has emerged as a robust platform for disease modeling and developing innovative treatments for genetic cardiac diseases. The review finds that PE, when combined with iPSC-CMs, holds significant promise for treating genetic cardiac diseases by addressing their root causes. This approach could revolutionize personalized medicine, offering more effective and precise treatments. Future research should focus on refining these technologies and their clinical applications.

Advancing CRISPR base editing technology through innovative strategies and ideas.

The innovation of CRISPR/Cas gene editing technology has developed rapidly in recent years. It is widely used in the fields of disease animal model construction, biological breeding, disease diagnosis and screening, gene therapy, cell localization, cell lineage tracking, synthetic biology, information storage, etc. However, developing idealized editors in various fields is still a goal for future development. This article focuses on the development and innovation of non-DSB editors BE and PE in the platform-based CRISPR system. It first explains the application of ideas for improvement such as "substitution", "combination", "adaptation", and "adjustment" in BE and PE development and then catalogues the ingenious inversions and leaps of thought reflected in the innovations made to CRISPR technology. It will then elaborate on the efforts currently being made to develop small editors to solve the problem of AAV overload and summarize the current application status of editors for in vivo gene modification using AAV as a delivery system. Finally, it summarizes the inspiration brought by CRISPR/Cas innovation and assesses future prospects for development of an idealized editor.

Precise Insertion of AttB Sequences in Goat Genome Using Enhanced Prime Editor.

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3' terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals.

Efficient Rescue of Retinal Degeneration in Pde6a Mice by Engineered Base Editing and Prime Editing.

Retinitis pigmentosa (RP) is a complex spectrum of inherited retinal diseases marked by the gradual loss of photoreceptor cells, ultimately leading to blindness. Among these, mutations in PDE6A, responsible for encoding a cGMP-specific phosphodiesterase, stand out as pivotal in autosomal recessive RP (RP43). Unfortunately, no effective therapy currently exists for this specific form of RP. However, recent advancements in genome editing, such as base editing (BE) and prime editing (PE), offer a promising avenue for precise and efficient gene therapy. Here, it is illustrated that the engineered BE and PE systems, particularly PE, exhibit high efficiency in rescuing a target point mutation with minimal bystander effects in an RP mouse model carrying the Pde6a (c.2009A > G, p.D670G) mutation. The optimized BE and PE systems are first screened in N2a cells and subsequently assessed in electroporated mouse retinas. Notably, the optimal PE system, delivered via dual adeno-associated virus (AAV), precisely corrects the pathogenic mutation with average 9.4% efficiency, with no detectable bystander editing. This correction restores PDE6A protein expression, preserved photoreceptors, and rescued retinal function in Pde6a mice. Therefore, this study offers a proof-of-concept demonstration for the treatment of Pde6a-related retinal degeneration using BE and PE systems.

Extended pegRNAs enhance the editing capability of Prime editing.

Genome editing is highly valuable in biomedical research. Despite their versatility, current Prime editing (PE) techniques are limited to short sequence alterations [up to ~44 base pairs (bp)], and exhibit inconsistent or low efficiency across genomic loci, particularly when faced with poly-T sequences. To address these challenges, we developed an extended PE (exPE) technique that can potentially execute any precise genome editing. By harnessing RNA polymerase II (Pol II) promoters to transcribe extended PE guide RNAs (expegRNAs), exPE substantially improves editing efficiency and overcomes the challenges posed by poly-T sequences. Compared with conventional PE, exPE achieves up to a 14-fold increase in the efficiency of base conversions and short insertions, and, remarkably, up to a 259-fold improvement in regions with poly-T sequences. Uniquely, exPE enables seamless insertion of gene-sized DNA fragments into genomes, potentially correcting nearly 90% of human genetic variants, thereby broadening its applications in genetic research and therapy.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Efficient and precise genomic deletion in rice using enhanced prime editing.

Efficient and precise genomic deletion shows promise for investigating the function of proteins in plant research and enhancing agricultural traits. In this study, we tested the PRIME-Del (PDel) strategy using a pair of prime editing guide RNAs (pegRNAs) that targeted opposite DNA strands and achieved an average deletion efficiency of 55.8% for 60 bp fragment deletions at six endogenous targets. Moreover, as high as 84.2% precise deletion efficiency was obtained for a 2000 bp deletion at the OsGS1 site in transgenic rice plants. To add the bases that were unintentionally deleted between the two nicking sequences, we used the PDel/Syn strategy, which introduced multiple synonymous base mutations in the region that had to be patched in the RT template. The PDel/Syn strategy achieved an average of 58.1% deletion efficiency at six endogenous targets, which was higher than the PDel strategy. The strategies presented in this study contribute to achieving more accurate and flexible deletions in transgenic rice plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00153-9.

PE6c greatly enhances prime editing in transgenic rice plants.

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a-d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime-editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

Assembly of Baculovirus Vectors for Multiplexed Prime Editing.

Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.

Exploitation of enhanced prime editing for blocking aberrant angiogenesis.

INTRODUCTION: Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders. OBJECTIVES: The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy. METHODS: An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis. RESULTS: NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology. CONCLUSION: DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.

CRISPR/Cas-Mediated Genome Engineering in Plants: Application and Prospectives.

Genetic engineering has become an essential element in developing climate-resilient crops and environmentally sustainable solutions to respond to the increasing need for global food security. Genome editing using CRISPR/Cas [Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas)] technology is being applied to a variety of organisms, including plants. This technique has become popular because of its high specificity, effectiveness, and low production cost. Therefore, this technology has the potential to revolutionize agriculture and contribute to global food security. Over the past few years, increasing efforts have been seen in its application in developing higher-yielding, nutrition-rich, disease-resistant, and stress-tolerant "crops", fruits, and vegetables. Cas proteins such as Cas9, Cas12, Cas13, and Cas14, among others, have distinct architectures and have been used to create new genetic tools that improve features that are important for agriculture. The versatility of Cas has accelerated genomic analysis and facilitated the use of CRISPR/Cas to manipulate and alter nucleic acid sequences in cells of different organisms. This review provides the evolution of CRISPR technology exploring its mechanisms and contrasting it with traditional breeding and transgenic approaches to improve different aspects of stress tolerance. We have also discussed the CRISPR/Cas system and explored three Cas proteins that are currently known to exist: Cas12, Cas13, and Cas14 and their potential to generate foreign-DNA-free or non-transgenic crops that could be easily regulated for commercialization in most countries.

Emerging Perspectives on Prime Editor Delivery to the Brain.

Prime editing shows potential as a precision genome editing technology, as well as the potential to advance the development of next-generation nanomedicine for addressing neurological disorders. However, turning in prime editors (PEs), which are macromolecular complexes composed of CRISPR/Cas9 nickase fused with a reverse transcriptase and a prime editing guide RNA (pegRNA), to the brain remains a considerable challenge due to physiological obstacles, including the blood-brain barrier (BBB). This review article offers an up-to-date overview and perspective on the latest technologies and strategies for the precision delivery of PEs to the brain and passage through blood barriers. Furthermore, it delves into the scientific significance and possible therapeutic applications of prime editing in conditions related to neurological diseases. It is targeted at clinicians and clinical researchers working on advancing precision nanomedicine for neuropathologies.

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.

[Prime-Editing Methods and pegRNA Design Programs].

It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A ↔ G and C ↔ T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.

Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges.

As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, in vivo barriers, and the future landscape of the technology are discussed.

Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids").

Enrichment of prime-edited mammalian cells with surrogate PuroR reporters.

Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.