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A Manual for Genome and Transcriptome Engineering.
Genome and transcriptome engineering have emerged as powerful tools in modern biotechnology, driving advancements in precision medicine and novel therapeutics. In this review, we provide a comprehensive overview of the current methodologies, applications, and future directions in genome and transcriptome engineering. Through this, we aim to provide a guide for tool selection, critically analyzing the strengths, weaknesses, and best use cases of these tools to provide context on their suitability for various applications. We explore standard and recent developments in genome engineering, such as base editors and prime editing, and provide insight into tool selection for change of function (knockout, deletion, insertion, substitution) and change of expression (repression, activation) contexts. Advancements in transcriptome engineering are also explored, focusing on established technologies like antisense oligonucleotides (ASOs) and RNA interference (RNAi), as well as recent developments such as CRISPR-Cas13 and adenosine deaminases acting on RNA (ADAR). This review offers a comparison of different approaches to achieve similar biological goals, and consideration of high-throughput applications that enable the probing of a variety of targets. This review elucidates the transformative impact of genome and transcriptome engineering on biological research and clinical applications that will pave the way for future innovations in the field.
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Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity.
Existing CRISPR/cas-based biosensors usually improve sensitivity by target amplification, which is time-consuming and susceptible to impurities in complex biofluid. Herein, this is the first time a cascade CRISPR/cas (casCRISPR) system has been developed, which can provide a detection limit of 1.33 fM (∼1000 times lower than direct Cas13a-based miRNA detection) and single-base resolution for miR-17 detection without resorting to target amplification. casCRISPR can also be applied to detect miRNA in complicated cell extracts and serum samples. Overall, casCRISPR will provide a heuristic idea for CRISPR/cas based biosensing, and could be a promising tool for miRNA diagnostics.
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Quadruple pegRNA enables programmable and efficient large genomic insertion.
Precise, site-specific insertion of large gene sequences holds great promise for the treatment of diverse genetic disorders. Although prime editing using paired guide RNAs (pegRNAs) can mediate targeted integration, insertion efficiency drops sharply for payloads exceeding 300 base pairs1-3. Here we present a rationally designed quadruple pegRNA strategy (QuadPE) for efficient and programmable insertion of large DNA fragments. Through screening different designs, we identified that combinations of two genome-targeting pegRNAs in a PAM-out or PAM-in orientation, when paired with two donor-targeting pegRNAs in linear or circular form, yield optimal efficiency. Using QuadPE, we achieved stable integration efficiency of DNA fragments ranging from 1.6 to 26 kb, with efficiencies of around 40% at multiple loci with minimal off-target insertion activity. QuadPE substantially outperformed recombinase-mediated (PASSIGE and PASTE)4,5 and transposase-mediated (CAST)6 insertion systems, particularly for larger payloads, showing a 11-fold, 61-fold and 12-fold improvement for a 9.5 kb insertion, respectively. Notably, QuadPE was effective in both dividing and non-dividing primary cells such as human primary T cells and post-mitotic neurons, establishing QuadPE as a powerful and precise platform for large-fragment gene insertion without the need for double-stranded breaks or recombinases.
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Promoter editing for the genetic improvement of crops.
Gene expression plays a fundamental role in the regulation of agronomically important traits in crop plants. The genetic manipulation of plant promoters through genome editing has emerged as an effective strategy to create favorable traits in crops by altering the expression pattern of the pertinent genes. Promoter editing can be applied in a directed manner, where nucleotide sequences associated with favorable traits are precisely generated. Alternatively, promoter editing can also be exploited as a random mutagenic approach to generate novel genetic variations within a designated promoter, from which elite alleles are selected based on their phenotypic effects. Pioneering studies have demonstrated the potential of promoter editing in engineering agronomically important traits as well as in mining novel promoter alleles valuable for plant breeding. In this review, we provide an update on the application of promoter editing in crops for increased yield, enhanced tolerance to biotic and abiotic stresses, and improved quality. We also discuss several remaining technical bottlenecks and how this strategy may be better employed for the genetic improvement of crops in the future.
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Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation.
Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.
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Biosafety considerations triggered by genome-editing technologies.
Since the discovery of the double helix structure of deoxyribonucleic acid (DNA), significant progress has been made in engineering technologies such as gene analysis and editing, greatly promoting the development of biomedical research and clinical applications. In recent years, the rapid development of genome-editing technologies, especially the clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system, have brought unprecedented opportunities for biomedical research and clinical applications. However, with the widespread application of genome-editing technologies, biosafety issues have gradually attracted the attention of the scientific community and the public. Potential risks such as off-target effects, genomic instability, and ethical and legal issues need to be taken seriously, and their safety and effectiveness in clinical applications still require further verification. This review summarizes the current status and potential risks of gene editing technologies in the medical field and discusses how to ensure its safe application through policies, regulations, and technical means. Through in-depth exploration of these issues, we hope to provide a comprehensive perspective for the scientific community, policy makers, and the public to promote the safe and responsible application of genome-editing technologies.
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A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.
Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.
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Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review.
Various non-nucleic acid targets (ions, small molecules, polysaccharides, peptides/proteins/enzymes, cells, transcription factors) are important biomarkers. They play important roles in environmental protection, food safety and medical diagnosis. Therefore, it is necessary to detect non-nucleic acid targets from various samples before the situation deteriorates. Derived from prokaryotic immune systems, CRISPR/Cas tools have exhibited great promise in the field of biosensing, in addition to the well-known gene-editing function. However, most reported CRISPR/Cas-based biosensors are for nucleic acid detection and the application of non-nucleic acid targets is still in its infancy. To fully explore the potential of CRISPR/Cas-based biosensing systems, it is of great significance to summarize the strategies and prospects of CRISPR/Cas toolboxes in non-nucleic acid targets recognition. In this review, we introduced CRISPR/Cas systems and their characteristics in the field of detection. The progress of detecting six non-nucleic acid targets was outlined and reviewed based on CRISPR/Cas systems coupled with biotransduction elements, including aptamers, DNAzymes, riboswitches, enzymatic reactions, transcription factors, antigen-antibody interactions, allosteric probes, in vitro transcription processes, steric hindrance effectors, etc. The development challenges and prospects in this field were also put forward. As such, this comprehensive review would provide valuable information for the expansion of the powerful CRISPR/Cas toolboxes into multiple detection fields.
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Optimizations of Cas12a- and Cas12i-based adenine base editors for efficient precision editing in the plant genome.
A strategy coupling high-activity nucleases with dimeric TadA-8e optimizes plant Cas12-based adenine base editors, boosts editing efficiency, and provides precise editors for crop breeding and genomics research.
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Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo.
Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge. Here we report prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture. In two mouse models of genetic blindness, single injections of v3 PE-eVLPs resulted in therapeutically relevant levels of prime editing in the retina, protein expression restoration and partial visual function rescue. Optimized PE-eVLPs support transient in vivo delivery of prime editor ribonucleoproteins, enhancing the potential safety of prime editing by reducing off-target editing and obviating the possibility of oncogenic transgene integration.
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