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Non-nuclear localization signal-guided CRISPR/Cas9 ribonucleoproteins for translocation and gene editing via apoferritin delivery vectors.
Direct delivery of the Cas9/sgRNA ribonucleoprotein (RNP) via appropriate carriers has been proved to be an important advance for the in vivo translocation and gene editing of CRISPR/Cas9. These carriers often require the nuclear localization signal (NLS) to fuse with Cas9 or the NLS-bearing protein to form a complex with Cas9 to enter the nucleus. In this study, we introduced apoferritin nanocages as carriers and DOX as a nuclear trigger for the nuclear transport of the Cas9/sgRNA ribonucleoprotein without the NLS (RNP-). Our experiments showed that loading RNP- and DOX into 4L-FTH subunit-based apoferritin nanocages leads to efficient endocytosis and lysosomal escape. Specifically, when DOX was administered at a concentration of 1 μM, we observed the activation of cellular defense mechanisms, which effectively facilitated the translocation of 4L-HFn@RNP-/DOX nanoparticles into the nucleus, thereby enabling intranuclear RNP- delivery. This strategy has been empirically demonstrated to achieve gene editing efficiencies of approximately 33% for the Lcn2 gene in MDA-MB-231 cells and 17.9% for the copepod green fluorescent protein (copGFP) gene in HeLa.copGFP cells in vitro. Moreover, in vivo editing efficacy, as tested in a HeLa.copGFP nude mouse model, was confirmed to be 16%. This delivery system presents a novel therapeutic approach for the nuclear delivery of small molecules or nucleic acid drugs, potentially overcoming the challenges associated with nuclear entry barriers.
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In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
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Prime Editing: Mechanism and Developments.
Prime editing is a "search and replace" based genome editing method that can introduce point mutations, insertions, and deletions into DNA at desired sites, making it the editing technique capable of making the most diverse range of edits. Prime editing utilizes a Cas9 nickase fused to a reverse transcriptase to introduce edits encoded by a specially designed DNA oligonucleotide called a prime editing guide RNA (pegRNA). While prime editing has been shown to achieve robust editing in different genomic loci and in different cell types, this technology still has the potential for further optimization and improvement. Many groups have been successful in improving prime editing outcomes by optimizing the design of prime editors and pegRNAs. Additionally, modulating key cellular pathways has also been an effective tool for achieving high levels of editing. Due to its high efficiency and relative safety, prime editing has the potential to transform disease modeling and genomic medicine.
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CRISPR/Cas Technologies Applied to Pseudogenes.
Pseudogenes have been considered as nonfunctional copies of their parental genes for a long time. Indeed, they have been often defined "junk DNA" or "transcriptional noise." However, with the identification of their involvement in several biological processes, the necessity of their study is inevitably growing up. The manipulation of pseudogene expression is complicated by their high homology with parental genes and by the fact that most of them work at the transcriptional level as noncoding RNAs. With the advent of CRISPR/Cas technology, these problems can be overcome. Particularly, as we describe in this chapter, it is possible: To perform genome editing, obtaining the complete elimination of the pseudogene genomic sequence (knock-out), preventing pseudogene transcription, introducing specific mutations in the pseudogene sequence, or introducing a specific sequence (knock-in). To positively or negatively manipulate pseudogene transcription. To target pseudogene RNA and negatively regulate its expression. To edit pseudogene DNA and RNA and alter a specific sequence. Moreover, CRISPR/Cas technology can be used as an RNA Binding Protein system for molecular biology techniques (such as RNA immunoprecipitation and pull-down), as well as for transcript tracking and live imaging.
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Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.
Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency hold a significant risk of severe hemolytic crises under oxidative stress. Currently, the definitive and curative treatment for the disorder has not been developed. Among over 200 G6PD variants, G6PD Viangchan (c.871 G > A) is the most prevalent and has been extensively studied in Southeast Asia. This study assessed the effectiveness of prime editing for correcting the G6PD Viangchan mutation in an established mutant HEK293T cell line and G6PD-deficient induced pluripotent stem cells (iPSCs). Using optimized modalities, prime editing achieved a high correction efficiency of over 25% in the HEK293T cells. In iPSCs, this gene editing tool yielded satisfactory correction outcomes, with approximately 5% corrected alleles. Our findings indicate that prime editing provides high precision, producing minimal by-products below baseline and showing undetectable off-target effects. Overall, prime editing has the potential to correct the G6PD Viangchan mutation, providing a valuable approach for future therapeutic strategies and the generation of isogenic cell lines to promote extensive studies in drug discovery and the pathogenesis of the G6PD variant.
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PEGylated Chitosan for Nonviral Aerosol and Mucosal Delivery of the CRISPR/Cas9 System in Vitro.
Chitosan has been widely employed to deliver nucleic acids such as siRNA and plasmids. However, chitosan-mediated delivery of a gene-editing system has not been reported yet. In this study, poly(ethylene glycol) monomethyl ether (mPEG) was conjugated to chitosan with different molecular weights (low molecular weight and medium molecular weight chitosan) achieving a high degree of substitution as identified by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) spectra. PEGylated chitosan/pSpCas9-2A-GFP nanocomplexes were formed at different N/ P (amine group to phosphate group) ratios and characterized in terms of size and zeta potential. The nanocomplexes developed showed the capability to protect loaded nucleic acids from DNase I digestion and from the stresses of nebulization. In addition, we demonstrated that the PEG conjugation of chitosan improved the mucus-penetration capability of the formed nanocomplexes at N/ P ratios of 5, 10, 20, and 30. Finally, PEGylated low molecular weight chitosan nanocomplexes showed optimal transfection efficiency at an N/ P ratio of 20, while PEGylated medium molecular weight chitosan nanocomplexes showed an optimal transfection efficiency at an N/ P ratio of 5 at pH 6.5 and 6.8. This study established the basis for the delivery of a gene-editing system by PEGylated chitosan nanocomplexes.
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Genome Editing of C. elegans.
Caenorhabditis elegans, a 1 mm long free-living nematode, is a traditional model animal for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle, well-annotated genome, simple morphology, and transparency. Recently, genome editing technologies have been increasingly used in C. elegans, thereby facilitating their genetic analyses. Here, I introduce a protocol frequently used in C. elegans genome editing.
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A three-plasmid-containing CRISPR-Cas9 platform to engineer Bacillus velezensis 916 as an efficient biocontrol agent.
Bacillus velezensis (Bv) is a widely used biocontrol agent against plant diseases, mainly because its genome contains numerous non-ribosomal peptide synthetases (NRPS) gene clusters for the synthesis of various cyclic lipopeptides (CLPs). The domesticated strain Bv916, capable of co-producing four CLPs, has been successfully applied for green control of rice sheath blight and angular leaf spot. To enhance Bv916's biological control efficacy while maintaining environmental safety, it is essential to establish a food-grade gene editing platform in Bv916. Here, a three-plasmid CRISPR-Cas9 platform for Bv916 was constructed using the thermosensitive origin pET194ts, constitutive P43 promoters for Cas9, the specific promoter Psrf for single guide RNAs (sgRNAs), and three resistance gene expression cassettes. By replacing the native promoters of ComX and RecA in Bv916 with the strong promoters P43 and PrepU, respectively, this platform achieved a single-gene editing efficiency of 96%, while the simultaneous dual-gene editing efficiency reached 61%, with each round completed within five business days. Furthermore, this gene editing platform is used to replace promoters of four NRPS gene clusters (loc, srf, bl, and fen) in Bv916 with strong constitutive promoters (PB, PA, P43, and PrepU), generating the derivative BvLSBF. Compared to Bv916, BvLSBF showed 6.8-fold, 5.9-fold, 10.9-fold, and 6.2-fold increases in locillomycin, surfactin, bacillomycin L, and fengycin, respectively. Its antagonistic activity against plant pathogens was also significantly enhanced. This system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors, offering significant potential for sustainable agriculture.IMPORTANCEIn this study, a food-grade three-plasmid CRISPR-Cas9 platform for Bv916 was established by incorporating the optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors. This gene editing system was used to achieve gene insertion, deletion, and replacement in Bv916, particularly by editing four non-ribosomal peptide synthetase (NRPS) gene clusters. This resulted in increased production of four cyclic lipopeptides and significantly enhanced antibacterial and antifungal activity.
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Optimized protocol for efficient generation, confirmation, transformation, and CRISPR editing of grapevine hairy roots.
Hairy root cultures (HRCs) are powerful tools in plant biotechnology but show variable establishment efficiencies, limiting broader applications. Here, we present a standardized and optimized reference methodology for the routine generation, multiplication, and maintenance of HRCs across diverse grapevine genotypes. Our workflow evaluated three Rhizobium strains, seven grapevine cultivars (three Vitis vinifera cultivars; four Vitis rootstock hybrids), multiple explant types, infection protocols, co-cultivation times, growth media types, and anti-browning agents. The resulting protocol was effective for all grapevine genotypes and, with minor adjustments, also yielded HRCs from two other important South African plant species, namely Sutherlandia frutescens and Aspalathus linearis. Useful molecular tools were developed for transformation and selection of HRCs, including universal multiplex primers for confirmation of transformation, tested antibiotic resistance markers (kanamycin and hygromycin), and fluorescent reporters (DsRed and eyGFPuv), with DsRed found to be particularly versatile. To test the system, we overexpressed the VviMYBA1 transcription factor gene, leading to increased anthocyanin accumulation and red pigmentation in HRCs. Additionally, we achieved CRISPR/Cas9 editing of the VviPUB19 gene, the first report of CRISPR-edited grapevine HRCs. Gene editing combined with HRCs can facilitate rapid gene function studies, offering an efficient alternative or pre-screening system to whole-plant transformations, that could support advanced functional genomics and biotechnological applications in grapevine.
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Commentary on "CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice".
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