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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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Effective gene editing of melanoma by delivery of Cas9 mRNA with highly branched poly(β-amino ester).
Melanoma, the most aggressive form of skin cancer, still lacks clinically reliable treatments. CRISPR/Cas9 gene editing offers a promising approach for melanoma therapy; however, the efficient and safe delivery of CRISPR/Cas9 components to melanoma cells remains a formidable challenge. Here, we report the development of highly branched poly(β-amino ester) (HPAE) for the efficient delivery of Cas9 mRNA to melanoma cells and for transdermal application in vivo. HPAE is synthesized via a one-pot "A2 + B4 + C2" type Michael addition strategy and is capable of efficiently condensing mRNA to form nanosized polyplexes with a moderate positive charge. In human embryonic kidney cells (HEK293T), mouse melanoma cells (B16F10), and human melanoma cells (A2058 and A375), up to 76.3%, 74.6%, 16.5%, and 57.1% mRNA transfection efficiency was achieved without obvious cytotoxicity. Moreover, repeated dosing further enhanced mRNA transfection efficiency, resulting in sustained gene expression. Importantly, HPAE enables the effective co-delivery of Cas9 mRNA and sgRNA to melanoma cells, facilitating up to 25.5% gene knockout. HPAE also demonstrates strong transdermal capability, resulting in high in vivo mRNA expression following direct topical administration. This study demonstrates the feasibility of treating melanoma through the transdermal application of the CRISPR/Cas9 system, enabled by the efficient mRNA delivery provided by HPAE.
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Methods for characterizing, applying, and teaching CRISPR-Cas systems.
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Exploiting epigenetics for the treatment of inborn errors of metabolism.
Gene therapy is currently considered as the optimal treatment for inborn errors of metabolism (IEMs), as it aims to permanently compensate for the primary genetic defect. However, emerging gene editing approaches such as CRISPR-Cas9, in which the DNA of the host organism is edited at a precise location, may have outperforming therapeutic potential. Gene editing strategies aim to correct the actual genetic mutation, while circumventing issues associated with conventional compensation gene therapy. Such strategies can also be repurposed to normalize gene expression changes that occur secondary to the genetic defect. Moreover, besides the genetic causes of IEMs, it is increasingly recognized that their clinical phenotypes are associated with epigenetic changes. Because epigenetic alterations are principally reversible, this may offer new opportunities for treatment of IEM patients. Here, we present an overview of the promises of epigenetics in eventually treating IEMs. We discuss the concepts of gene and epigenetic editing, and the advantages and disadvantages of current and upcoming gene-based therapies for treatment of IEMs.
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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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Computer-aided engineering of CRISPR-Cas proteins for enhanced human genome editing.
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Applications of Functional Genomics for Drug Discovery.
Many diseases, such as diabetes, autoimmune diseases, cancer, and neurological disorders, are caused by a dysregulation of a complex interplay of genes. Genome-wide association studies have identified thousands of disease-linked polymorphisms in the human population. However, detailing the causative gene expression or functional changes underlying those associations has been elusive in many cases. Functional genomics is an emerging field of research that aims to deconvolute the link between genotype and phenotype by making use of large -omic data sets and next-generation gene and epigenome editing tools to perturb genes of interest. Here we review how functional genomic tools can be used to better understand the biological interplay between genes, improve disease modeling, and identify novel drug targets. Incorporation of functional genomic capabilities into conventional drug development pipelines is predicted to expedite the development of first-in-class therapeutics.
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A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.
Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.
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Protocol for genomic editing in human resting primary NK cells and NK-92 cells via CRISPR-Cas9 ribonucleoproteins.
Here, we present a protocol to perform CRISPR-Cas9 genome editing in human resting primary natural killer (NK) and NK-92 cells. We describe steps for guide RNA selection, ribonucleoprotein (RNP) complex formation, delivery via Nucleofection, and analysis of CRISPR edits to assess editing efficiencies. This protocol offers a tool for functional studies in NK cells, paving the way for potential applications in immunotherapy and beyond. We also discuss limitations such as off-target effects and cell-type-specific considerations.
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Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing.
One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.
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