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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Unleashed: Transforming Gene Editing With Breakthroughs, Applications, and Ethical Dilemmas.
The most significant breakthrough in gene editing is the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. This innovative technology enables scientists to insert or delete genes using specific enzymes, facilitating modifications to genomes that can influence an organism's phenotype. The Cas9 enzyme is the most widely used within the CRISPR framework and has already received approval for treating sickle cell disease, with many other applications likely to follow. As this rapidly evolving field continues to advance, it holds great promise for addressing genetic disorders and diseases. This article will explore the various enzymes available in the CRISPR system, the range of diseases and conditions that could be treated using this technology, alternative gene therapy methods, and the ethical considerations surrounding its use.
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An adenine base editor with expanded targeting scope using SpCas9-NGv1 in rice.
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A novel cloning strategy for one-step assembly of multiplex CRISPR vectors.
One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.
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Successful In Vitro Modification of the Dmd Gene Using Prime Editing.
Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5'-TTCT-3' motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.
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Current understanding of mitochondrial DNA genetic diseases and gene therapy.
Mitochondria, as crucial organelles within eukaryotic cells, have their proteins and RNAs encoded by both the nuclear genome and the mitochondrial genome. They play vital roles in energy regulation, cellular metabolism, signal transduction, and various other physiological activities. Additionally, mitochondria interact with multiple organelles to collectively maintain cellular homeostasis. Mitochondria can also be transferred between cells and tissues through mechanisms such as migrasomes. Mitochondrial DNA (mtDNA) mutations often cause severe inherited rare diseases, characterized by tissue specificity, heterogeneity, multiple mutation sites, and challenges in achieving a complete cure. Gene editing of mtDNA holds promise for fundamentally curing such diseases. Traditional gene-editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), as well as novel gene editors like DddA-derived cytosine base editors (DdCBEs), have been demonstrated to correct certain mtDNA mutations. However, CRISPR-based technologies-despite their superior programmability and efficiency-are currently limited due to the technical bottleneck of inefficient sgRNA delivery into mitochondria. This article systematically reviews the structure and function of mitochondria, related diseases, and the current state of mtDNA gene-editing therapies. Furthermore, it explores future directions for optimizing therapeutic tools to overcome the challenge of sgRNA delivery, thereby addressing the treatment barriers posed by pathogenic mtDNA mutations in inherited rare diseases.
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Prime editing: evolution of CRISPR-Cas system for a robust next-generation genome editing in plants.
Prime editing is a propulsive and versatile genome engineering technology that enables precise installation of all possible 12 base-to-base conversions, targeted insertions, deletions, and combinatorial modifications without inducing double-strand break (DSB) or requiring exogenous donor DNA template. Since its inception, prime editing has been rapidly adopted across plant systems, offering a powerful platform for functional genomics, trait improvement, and precision molecular breeding. This review comprehensively traces the evolution of prime editors (PEs) from first-generation PE1 to advanced variants such as PE7 and TwinPE. We detail the key technological milestones, including innovations in protein engineering, prime editing guide RNA (pegRNA) architectural improvement, and strategic modulation of host DNA repair mechanisms aimed at enhancing editing efficiency, precision, and versatility. Further, we provide an in-depth overview of plant-adapted prime editing systems, focusing on codon optimization, promoter refinement, pegRNA scaffold engineering, and the integration of plant-compatible Cas9 and reverse transcriptase variants. Special emphasis is given to the application of prime editing in diverse crop species. By consolidating recent advances and highlighting emerging trends, this review presents a forward-looking perspective on the deployment of prime editors (PEs) as transformative tool for precision genome engineering and sustainable crop improvement.
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Guanidyl-rich highly branched poly(β-amino ester)s for the delivery of dual CRISPR ribonucleoprotein for efficient large DNA fragment deletion.
Gene editing technologies, particularly clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, have revolutionized the ability to modify gene sequences in living cells for therapeutic purposes. Delivery of CRISPR/Cas ribonucleoprotein (RNP) is preferred over its DNA and RNA formats in terms of gene editing effectiveness and low risk of off-target events. However, the intracellular delivery of RNP poses significant challenges and necessitates the development of non-viral vectors. Our previous study has demonstrated that phenyl guanidine (PG) group modified linear poly(β-amino ester)s (PAEs) can facilitate CRISPR/Cas9 RNP mediated gene knockout in HeLa cells. Here, we further investigated the utilization of highly branched PAEs (HPAEs) with PG groups (HPAE-PG) for efficient delivery of cytosolic protein and CRISPR/Cas9 RNP complexes, while also examining the influence of branching units and branching ratios on the delivery process. The efficiency of HPAE-PG/RNP transfection for large DNA fragment deletion was assessed using a dual sgRNA-guided approach to delete exon 80 of the human COL7A1 gene, which harbors mutations associated with dystrophic epidermolysis bullosa (DEB). Our findings demonstrate that HPAE-PG/RNP successfully induced a deletion of 56 base pairs (exon 80) within COL7A1 in both HEK cells and keratinocytes derived from recessive DEB patients. This study highlights the potential of HPAE-PG as a non-viral vector for large DNA fragment deletion, emphasizing the importance of branching factors of HPAEs in optimizing CRISPR RNP delivery for therapeutic applications in genetic disorders.
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The Design and Application of DNA-Editing Enzymes as Base Editors.
DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases.
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CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.
The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.
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Advanced peptide nanoparticles enable robust and efficient delivery of gene editors across cell types.
Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a major challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the system's versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents an important advancement in the feasibility and scalability of gene-editing delivery technologies.
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