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Compositional Optimization of CRISPR/Cas9 Lipid Nanoparticles for Efficient Knockdown of Target Genes.
Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.
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CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines.
CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.
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Therapeutic Strategies of Clustered Regularly Interspaced Palindromic Repeats-Cas Systems for Different Viral Infections.
The clustered regularly interspaced palindromic repeats (CRISPR)/Cas systems have rapidly transitioned from intriguing prokaryotic defense systems to an efficient therapeutic tool. This cutting-edge technology is currently revolutionizing to combat hostile viruses because of its reproducibility, high potency, ease of use, limited off-target activity, and development of quick immune response against viruses. CRISPR gene editing technology eliminates the virus by breaking the DNA that ultimately halts viral replication. This review summarizes the advancements that have been made in the use of CRISPR-Cas technology in viral therapeutics.
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Emerging trends in prime editing for precision genome editing.
Prime editing is an advanced genome editing technology that enables precise genetic modifications without inducing double-strand breaks or requiring donor DNA templates. Prime editing has rapidly become a versatile tool, supporting a wide range of genetic modifications, including point mutations, insertions and deletions. Here we examine the evolution of prime editing technologies, detailing advancements from the initial prime editing systems to recent innovations that enhance editing efficiency. Through structural modifications and improved delivery methods, prime editing has expanded its applicability across eukaryotic systems. By enabling access to previously challenging mutations, prime editing opens new avenues for therapeutic development and precision genetic research, with efficiency, specificity and accessibility expected to shape its future impact in genome engineering.
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Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.
BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.
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Inverse pharmacology: Approaches and tools for introducing druggability into engineered proteins.
A major feature of twenty-first century medical research is the development of therapeutic strategies that use 'biologics' (large molecules, usually engineered proteins) and living cells instead of, or as well as, the small molecules that were the basis of pharmacology in earlier eras. The high power of these techniques can bring correspondingly high risk, and therefore the need for the potential for external control. One way of exerting control on therapeutic proteins is to make them responsive to small molecules; in a clinical context, these small molecules themselves have to be safe. Conventional pharmacology has resulted in thousands of small molecules licensed for use in humans, and detailed structural data on their binding to their protein targets. In principle, these data can be used to facilitate the engineering of drug-responsive modules, taken from natural proteins, into synthetic proteins. This has been done for some years (for example, Cre-ERT2) but usually in a painstaking manner. Recently, we have developed the bioinformatic tool SynPharm to facilitate the design of drug-responsive proteins. In this review, we outline the history of the field, the design and use of the Synpharm tool, and describe our own experiences in engineering druggability into the Cpf1 effector of CRISPR gene editing.
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Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.
We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.
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A non-cationic nucleic acid nanogel for the delivery of the CRISPR/Cas9 gene editing tool.
Herein, we report a non-cationic DNA-crosslinked nanogel for intracellular delivery of a Cas9 and single guide RNA (Cas9/sgRNA) complex. A DNA-grafted polycaprolactone brush (DNA-g-PCL) is first loaded with the Cas9/sgRNA complex and then crosslinked by DNA linkers via nucleic acid hybridization to form a nanosized hydrogel, in which the gene editing tools are embedded and protected inside. With compact architecture, the Cas9/sgRNA complex-containing nanogel exhibited excellent physiological stability against nuclease digestion and enhanced cellular uptake efficiency, making the delivery system a promising tool for target genome editing.
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Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.
Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5'-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI857 and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simultaneous and accurate editing of these two genes with an efficiency of 30%. This enabled the edited cells to retain their lysogenic state at 42 °C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNA method holds significant potential for widespread and practical use in synthetic biology.
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Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system.
Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.
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