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Artificial Intelligence in CRISPR-Cas Systems: A Review of Tool Applications.
Genetic engineering is a method used to alter an organism's DNA, which could entail altering a base pair, removing a section of DNA, or introducing a new DNA segment. Over time, genetic engineering has progressed from basic cloning for research purposes to advanced synthetic biology, leading to new biomedical applications. Targeted genomic editing is one method of cellular reprogramming that aims to change the state of a cell. The invention of CRISPR Cas systems has greatly simplified gene editing. These systems use a unique RNA-guided DNA endonuclease, a protein that can cut DNA and be trained to target new places by changing the sequence of its guide RNA. Integrating CRISPR-Cas systems with artificial intelligence opens new insights into the study of genetic engineering and its applications. Extensive research utilizing deep learning and machine learning has been conducted to predict the outcomes of CRISPR-Cas9 editing. Artificial intelligence also predicts RNA editing events and CRISPR off-target cleavage sites. Scientists often struggle to identify the ideal perturbation for their specific application because of the ample search space and expensive genetic trials. The algorithmic method using artificial intelligence utilizes the cause-and-effect link between variables in a complicated system like genome regulation to determine which perturbation is most effective in each successive round of testing, thereby making artificial intelligence an effective technique in gene editing.
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Delivery of nucleic acid based genome editing platforms via lipid nanoparticles: Clinical applications.
CRISPR/Cas technology presents a promising approach for treating a wide range of diseases, including cancer and genetic disorders. Despite its potential, the translation of CRISPR/Cas into effective in-vivo gene therapy encounters challenges, primarily due to the need for safe and efficient delivery mechanisms. Lipid nanoparticles (LNPs), FDA-approved for RNA delivery, show potential for delivering also CRISPR/Cas, offering the capability to efficiently encapsulate large mRNA molecules with single guide RNAs. However, achieving precise targeting in-vivo remains a significant obstacle, necessitating further research into optimizing LNP formulations. Strategies to enhance specificity, such as modifying LNP structures and incorporating targeting ligands, are explored to improve organ and cell type targeting. Furthermore, the development of base and prime editing technology presents a potential breakthrough, offering precise modifications without generating double-strand breaks (DSBs). Prime editing, particularly when delivered via targeted LNPs, holds promise for treating diverse diseases safely and precisely. This review assesses both the progress made and the persistent challenges faced in using LNP-encapsulated CRISPR-based technologies for therapeutic purposes, with a particular focus on clinical translation.
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Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells.
CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs.
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[Research advances in photoactivatable CRISPR gene editing technology].
Since the emergence of CRISPR/Cas9, gene editing technologies have attracted increasing attention, in particular type II systems, in which nucleases consist of only a single protein. The effectors include type II Cas9, type V Cas12 and type VI Cas13, which allow precise genomic DNA or RNA editing. Catalytically inactive CRISPR/Cas9 can also be used as a platform to recruit effectors such as transcription factors, epigenetic factors, and/or base modification enzymes to target gene loci. On the other hand, optogenetics offers spatial, temporal, and reversible control of biological processes. CRISPR and optogenetics can enable precise gene editing in vitro and in vivo at the spatiotemporal level, which has a broad applicability in biology and medicine. This article has provided a review for the research advance in photoactivatable CRISPR systems, with details for the design and application of such tools and a discussion over the limitations of the current methods, which may shed light on this emerging field.
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AAV-mediated gene therapies by miniature gene editing tools.
The advent of CRISPR-Cas has revolutionized precise gene editing. While pioneering CRISPR nucleases like Cas9 and Cas12 generate targeted DNA double-strand breaks (DSB) for knockout or homology-directed repair, next generation CRISPR technologies enable gene editing without DNA DSB. Base editors directly convert bases, prime editors make diverse alterations, and dead Cas-regulator fusions allow nuanced control of gene expression, avoiding potentially risks like translocations. Meanwhile, the discovery of diminutive Cas12 orthologs and Obligate Mobile Element-Guided Activity (OMEGA) nucleases has overcome cargo limitations of adeno-associated viral vectors, expanding prospects for in vivo therapeutic delivery. Here, we review the ever-evolving landscape of cutting-edge gene editing tools, focusing on miniature Cas12 orthologs and OMEGA effectors amenable to single AAV packaging. We also summarize CRISPR therapies delivered using AAV vectors, discuss challenges such as efficiency and specificity, and look to the future of this transformative field of in vivo gene editing enabled by AAV vectors delivery.
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Base editing and nanoparticle transfection of airway cell types essential for treatment of cystic fibrosis.
Cystic fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucus impairing the airway epithelia. Durable correction of these variants in airway cells remains a therapeutic challenge for about 10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant, 3120+1G>A, was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types resulting in notable enrichment of CFTR-expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited, but they decreased as a fraction of total cells and CFTR-expressing cells compared with unedited cells. CRISPR base editors delivered by polymeric nanoparticles (PNPs) facilitated functional rescue of CFTR to clinically meaningful levels in immortalized and primary airway cells. PNPs delivered GFP-encoding RNA to progenitor airway cells in fully differentiated airway cultures. Vitronectin was a major component of the PNP corona that formed in vivo, but preincubation with vitronectin did not enhance delivery. Together, these findings validate a scalable, nonviral platform with compelling translational promise for treating CF and other respiratory diseases involving respiratory epithelial cell dysfunction.
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Recent advances in stem cells and gene editing: Drug discovery and therapeutics.
The recently introduced genome editing technology has had a remarkable impact on genetic medicine. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas nucleases are the three major platforms used for priming of stem cells or correction of mutated genes. Among these nucleases, CRISPR/Cas is the most easily applicable. Various CRISPR/Cas variants such as base editors, prime editors, mad7 nucleases, RESCUE, REPAIR, digenome sequencing, and SHERLOCK are being developed and considered as a promising tool for gene therapy and drug discovery. These advances in the CRISPR/Cas platform have enabled the correction of gene mutations from DNA to RNA level and validation of the safety of genome editing performance at a very precise level by allowing the detection of one base-pair mismatch. These promising alternatives of the CRISPR/Cas system can benefit millions of patients with intractable diseases. Although the therapeutic effects of stem cells have been confirmed in a wide range of disease models, their safety still remains an issue. Hence, scientists are concentrating on generating functionally improved stem cells by using programmable nucleases such as CRISPR. Therefore, in this chapter, we have summarized the applicable options of the CRISPR/Cas platforms by weighing their advantages and limitations in drug discovery and gene therapy.
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A simple, quick, and efficient CRISPR/Cas9 genome editing method for human induced pluripotent stem cells.
Induced pluripotent stem cells (iPSCs) have become an essential research platform to study different human diseases once being discovered by Dr. Shinya Yamanaka in 2006. Another breakthrough in biomedical research is the application of CRISPR/Cas9 system for genome editing in mammalian cells. Although numerous studies have been done to develop methods for gene editing in iPSCs, the current approaches suffer from several limitations, including time and labor consuming, low editing efficiency, and potential off-target effects. In the current study, we report an electroporation-mediated plasmid CRISPR/Cas9 delivery approach for genome editing in iPSCs. With this approach, an edited iPSC cell line could be obtained within 2 weeks. In addition, the transit introducing of CRISPR/Cas9 machinery could minimize genomic integration of Cas9 gene, which avoided potential long-term side effects of Cas9 enzyme. We showed that CRISPR/Cas9-mediated genomic editing did not affect pluripotency and differentiation ability of iPSCs. With the quickly evolving of both iPSC and CRISPR/Cas9-mediated genome editing research fields, we believe that our method can significantly facilitate the application of genome editing in iPSCs research.
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Developing a highly efficient and wildly adaptive CRISPR-SaCas9 toolset for plant genome editing.
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Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor.
Rhodotorula toruloides is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in R. toruloides to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in R. toruloides. The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.
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