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Enhanced CRISPR-Cas9 RNA system delivery using cell penetrating peptides-based nanoparticles for efficient in vitro and in vivo applications.
CRISPR-Cas9 system has emerged as a revolutionary gene-editing tool with huge therapeutic potential for addressing the underlying genetic causes of various diseases, including cancer. However, there are challenges such as the delivery method that must be overcome for its clinical application. In addition to the risk of nuclease degradation and rapid clearance of the CRISPR-Cas9 system by macrophages, the large size of Cas9, the high anionic charge density and hydrophilic nature of the RNA hinder their intracellular delivery and overall gene transfection efficiency. In this study, we engineered a novel Peptide-Based Nanoparticles ADGN for the delivery of long RNA. ADGN peptides can form stable self-assembled nanoparticles with CRISPR-Cas9 RNA. They have the ability to cross the cell membrane of various cell types, exhibiting a preference for cancer cells that overexpress laminin receptor and safeguard RNA prior their delivery into the cytoplasm. We demonstrate that ADGN peptides significantly promote CRISPR-Cas9 mediated knockout of the luciferase gene in vitro achieving 60 % efficiency with a preference for G insertion at the targeted site of luciferase gene. Moreover, we have provided evidence that these nanoparticles can also be systemically intravenously administrated in vivo in mice to deliver a functional CRISPR-Cas9 system to tumoral lung cells orthotopically implanted in the mouse, resulting in an effective gene knockout in mice. We also demonstrated that the in vivo distribution of ADGN-RNA is influenced by its peptides to RNA molar ratio. This study introduces a promising new Peptide-Based Nanoparticles for delivering CRISPR-Cas9 system in its RNA form applicable in both in vitro and in vivo models.
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Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.
The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.
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PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation.
CRISPR technology holds significant promise for biological studies and gene therapies because of its high flexibility and efficiency when applied in mammalian cells. But endonuclease (e.g., Cas9) potentially generates undesired edits; thus, there is an urgent need to comprehensively identify off-target sites so that the genotoxicities can be accurately assessed. To date, it is still challenging to streamline the entire process to specifically label and efficiently enrich the cleavage sites from unknown genomic locations. Here we develop PEAC-seq, in which we adopt the Prime Editor to insert a sequence-optimized tag to the editing sites and enrich the tagged regions with site-specific primers for high throughput sequencing. Moreover, we demonstrate that PEAC-seq could identify DNA translocations, which are more genotoxic but usually overlooked by other off-target detection methods. As PEAC-seq does not rely on exogenous oligodeoxynucleotides to label the editing site, we also conduct in vivo off-target identification as proof of concept. In summary, PEAC-seq provides a comprehensive and streamlined strategy to identify CRISPR off-targeting sites in vitro and in vivo, as well as DNA translocation events. This technique further diversified the toolkit to evaluate the genotoxicity of CRISPR applications in research and clinics.
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Methods for creating transgenic primates.
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Overcoming barriers in CAR-NK immunotherapy: CRISPR-Driven advances in checkpoint editing and allogeneic design.
Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are emerging as an exciting avenue in cancer immunotherapy due to their potent cytotoxicity to malignant cells and lower risk of graft-versus-host disease (GvHD) than conventional T cell therapies. The new technology of CRISPR/Cas9 genome editing has significantly expedited the engineering of CAR-NK cells by enabling easy, multiplex, and precise changes to enhance their efficacy, persistence, and specificity to tumors. This review focuses on the incorporation of CRISPR technology into CAR-NK cell development. It examines uses of knockout of inhibitory checkpoint genes (CISH, PD-1, and TGFBR2), as well as knock-in of CAR into safe genomic locations and multiplex editing of CAR-NK cells to improve cytotoxicity against cancer while resisting suppression from the tumor microenvironment (TME). We further explore immuno-cytokine armoring strategies by knock-in of IL-15 or IL-12, to ensure prolonged proliferation and survival of NK cells, and investigate CRISPR-mediated knockouts of immune inhibitors like NKG2A and TIGIT, to evade immune strategies used by the tumor to evade immune destruction. Furthermore, CRISPR-mediated upregulation of the homing receptor enhances NK cell tumor infiltration, addressing a major obstacle in treating solid tumors. It is significant to mention the progress in generating off-the-shelf products, which is a key step supporting the pursuit of allogeneic therapies. While substantial progress has been made, challenges remain related to optimizing CRISPR delivery, off-target effects, and enhancing in vivo persistence. Future directions of CAR-NK studies will likely capitalize on next-generation genome editing tools and synthetic biology for the development of tunable and logic-gated CAR-NK cells. Overall, this review illustrates the revolutionary capacity of combining CRISPR technology with CAR-NK immunotherapy to develop next-generation programmable and efficacious treatments for hematologic and solid malignancies.
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Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.
A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.
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TIGR-Tas and the Expanding Universe of RNA-Guided Genome Editing Systems: A New Era Beyond CRISPR-Cas.
The recent discovery of TIGR-Tas (Tandem Interspaced Guide RNA-Targeting Systems) marks a major advance in the field of genome editing, introducing a new class of compact, programmable DNA-targeting systems that function independently of traditional CRISPR-Cas pathways. TIGR-Tas effectors use a novel dual-spacer guide RNA (tigRNA) to recognize both strands of target DNA without requiring a protospacer adjacent motif (PAM). These Tas proteins introduce double-stranded DNA cuts with characteristic 8-nucleotide 3' overhangs and are significantly smaller than Cas9, offering delivery advantages for in vivo editing. Structural analyses reveal homology to box C/D snoRNP proteins, suggesting a previously unrecognized evolutionary lineage of RNA-guided nucleases. This review positions TIGR-Tas at the forefront of a new wave of RNA-programmable genome-editing technologies. In parallel, I provide comparative insight into the diverse and increasingly modular CRISPR-Cas systems, including Cas9, Cas12, Cas13, and emerging effectors like Cas3, Cas10, CasΦ, and Cas14. While the CRISPR-Cas universe has revolutionized molecular biology, TIGR-Tas systems open a complementary and potentially more versatile path for programmable genome manipulation. I discuss mechanistic distinctions, evolutionary implications, and potential applications in human cells, synthetic biology, and therapeutic genome engineering.
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Microinjection in Zebrafish for Genome Editing and Functional Studies.
A major strength of zebrafish as a model organism is their rapid, in vitro development. The easy access to embryos compared to mammals, allows larval molecular and cellular composition to be manipulated by microinjection, providing a powerful avenue for biological and translational studies. Here, we describe the essential steps and different applications of microinjection in zebrafish for genome editing and functional studies, along with some experimental tips that are critical for microinjection success.
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Enhanced Genome Editing Tools For Multi-Gene Deletion Knock-Out Approaches Using Paired CRISPR sgRNAs in CHO Cells.
Since the establishment of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, powerful strategies for engineering of CHO cell lines have emerged. Nevertheless, there is still room to expand the scope of the CRISPR tool box for further applications to improve CHO cell factories. Here, the authors demonstrate activity of the alternative CRISPR endonuclease Cpf1 in CHO-K1 for the first time and that it can be used in parallel to CRISPR/Cas9 without any interference. Both, Cas9 and Cpf1, can be effectively used for multi-gene engineering with a strategy based on paired single guide RNAs (sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by removing the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. FUT8 also served as model to verify that deletion efficiency is size-independent (2-150 kb). To test the suitability for multi-gene approaches in combination with gene deletion, clones harboring triple deletions in β-1,4-Galactosyltransferase (B4GALT) isozymes are identified using solely conventional PCR/qPCR. In addition, two bicistronic transcription strategies are implemented to enable unequivocal pairing of sgRNAs: a CHO-derived tRNA linker that works for both, Cas9 and Cpf1, as well as paired sgRNAs in an array format, which can be used with Cpf1 due to its RNA processing ability. These strategies broaden the range of application of CRISPR for novel gene editing approaches in CHO cells and also enable the efficient realization of a genome-wide deletion library.
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CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1.
UNLABELLED: Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression. IMPORTANCE: Genetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800-1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains.
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