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Multigene editing: current approaches and beyond.
CRISPR/Cas9 multigene editing is an active and widely studied topic in the fields of biomedicine and biology. It involves a simultaneous participation of multiple single-guide RNAs (sgRNAs) to edit multiple target genes in a way that each gene is edited by one of these sgRNAs. There are possibly numerous sgRNA candidates capable of on-target editing on each of these genes with various efficiencies. Meanwhile, each of these sgRNA candidates may cause unwanted off-target editing at many other genes. Therefore, selection optimization of these multiple sgRNAs is demanded so as to minimize the number of sgRNAs and thus reduce the collective negative effects caused by the off-target editing. This survey reviews wet-laboratory approaches to the implementation of multigene editing and their needs of computational tools for better design. We found that though off-target editing is unavoidable during the gene editing, those disfavored cuttings by some target genes' sgRNAs can potentially become on-target editing sites for some other genes of interests. This off-to-on role conversion is beneficial to optimize the sgRNA selection in multigene editing. We present a preference cutting score to assess those beneficial off-target cutting sites, which have a few mismatches with their host genes' on-target editing sites. These potential sgRNAs can be prioritized for recommendation via ranking their on-target average cutting efficiency, the total off-target site number and their average preference cutting score. We also present case studies on cancer-associated genes to demonstrate tremendous usefulness of the new method.
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Therapeutic in vivo genome editing: innovations and challenges in rAAV vector-based CRISPR delivery.
The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorders. rAAV vectors have emerged as promising vehicles for in vivo gene therapy due to their favorable safety profile, high tissue specificity, and ability to induce sustained transgene expression. However, their limited packaging capacity has been a significant challenge for delivering large CRISPR molecules. To overcome this limitation, innovative strategies have been developed, including the use of compact Cas orthologs, dual rAAV vector systems, and trans-splicing rAAV vectors. These approaches have significantly improved the efficiency of genome editing for therapeutic applications. This review presents recent advancements in rAAV-CRISPR-mediated in vivo gene therapy, highlighting key technological innovations, current challenges, and the therapeutic potential of these strategies in the development of next-generation gene therapies.
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CRISPR-Cas9D10A Nickase-Assisted Genome Editing in Lactobacillus casei.
Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9D10A In addition to the P23-Cas9D10A and Pldh-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study.IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9D10A nickase-based genome editing in Lactobacillus casei, an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.
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Co-opting regulation bypass repair as a gene-correction strategy for monogenic diseases.
With the development of CRISPR-Cas9-mediated gene-editing technologies, correction of disease-causing mutations has become possible. However, current gene-correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene-correction strategy, co-opting regulation bypass repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans, which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair.
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Narrow path charted for editing genes of human embryos.
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Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis.
OBJECTIVES: Osteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects. METHODS: We performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1β and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1β. RESULTS: Loss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1β reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1β significantly mitigates the adverse effects of NGF blockade on the joints. CONCLUSIONS: CRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1β or IL-1β attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1β provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.
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Nanotechnological approaches for the targeted delivery of CRISPR-Cas systems for genomic modifications, biomolecular sensing, and precision medicine.
The integration strategies of CRISPR-Cas gene editing systems with nanotechnological approaches have achieved synergistic effects in targeting genes; correcting genetic disorders; and treating, sensing, and diagnosing a variety of cancers and metabolic, immunological, and complex infectious diseases-all having connectivity with distinct genetic cues and mutations. Numerous recent studies have demonstrated the use of the nano-scale properties of nanomaterials to tremendously improve the genomic-editing efficiencies of CRISPR/Cas systems for achieving 50% enhanced bioavailability, improved cell targetability, and gene-level specificity while minimizing immunogenicity, compared with conventional/ordinary delivery techniques. Thus, nano-delivery methods utilizing the unique properties of nanomaterials, molecular interactions, biocompatibility, targeted cellular uptake, and nuclear delivery capability effectively overcame the challenges of inefficient biomolecular delivery, and off-target effects were effectively overcome. Nano -carriers made up of materials such as DNA lattices, lipids, dendrimers, polymers, peptides, and metals (gold, silver, etc.) that were explored for facilitating the precise delivery of CRISPR/Cas components, sensing biomolecules, and diagnostic purposes are discussed in this review report. The ability of DNA scaffold materials to incorporate nano-CRISPR systems, to sense biomolecules, and for targeted cellular delivery of payloads (e.g., Cas9, Cas12, Cas13, and Cas14 proteins and single-guide RNAs (sgRNAs)) maximized gene targeting and improved therapeutic outcomes while achieving up to 90% efficiency compared with common/trivial delivery methods.
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Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.
CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.
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Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations.
Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.
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Biotechnological challenges: The scope of genome editing.
CRISPR/Cas9 can be considered as the biotechnological breakthrough of the century. Genome editing technologies have developed in a vertiginous way. While the genome editing of species, including animals, plants and bacteria has become a commonly used method, the application of CRISPR-Cas9 in human embryos has led to debates and interdisciplinary discussions. This brings multiple challenges for both scientists and those who must regulate the use of these techniques.
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