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Harnessing the evolving CRISPR/Cas9 for precision oncology.
The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating in CASGEVY™ approved for sickle cell anemia. Derived from a microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality in gene editing, has been harnessed as a versatile tool for precisely manipulating DNA in mammals. In the process of applying it to practice, the consecutive exploitation of novel orthologs and variants never ceases. It's conducive to understanding the essentialities of diseases, particularly cancer, which is crucial for diagnosis, prevention, and treatment. CRISPR/Cas9 is used not only to investigate tumorous genes functioning but also to model disparate cancers, providing valuable insights into tumor biology, resistance, and immune evasion. Upon cancer therapy, CRISPR/Cas9 is instrumental in developing individual and precise cancer therapies that can selectively activate or deactivate genes within tumor cells, aiming to cripple tumor growth and invasion and sensitize cancer cells to treatments. Furthermore, it facilitates the development of innovative treatments, enhancing the targeting efficiency of reprogrammed immune cells, exemplified by advancements in CAR-T regimen. Beyond therapy, it is a potent tool for screening susceptible genes, offering the possibility of intervening before the tumor initiative or progresses. However, despite its vast potential, the application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant efficacy, efficiency, technical, and safety considerations. Escalating technology innovations are warranted to address these issues. The CRISPR/Cas9 system is revolutionizing cancer research and treatment, opening up new avenues for advancements in our understanding and management of cancers. The integration of this evolving technology into clinical practice promises a new era of precision oncology, with targeted, personalized, and potentially curative therapies for cancer patients.
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A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing.
Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR-Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors.
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Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.
Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.
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Establishment of an efficient and PAM-relaxed LbCas12a genome editing tool in plants.
Cas12a is widely used in plant genome editing, but its targeting scope is constrained by stringent protospacer adjacent motif (PAM) requirements and variable activity across species, limiting its application at diverse genomic loci. LbCas12a-RRV-based editing system was established in nonheading Chinese cabbage, and T5exo-PF-LbCas12a was generated by introducing a triple mutation (D535G/S551F/D665N) and fusing with T5 exonuclease. This engineered system recognizes an expanded PAM sequence from 5'-VTTV-3' to 5'-NYHV-3'. The system exhibited efficient editing at noncanonical PAM sites in cabbage, tomato, and rice. Additionally, it successfully mediated large-fragment deletions via microhomology-mediated end joining (MMEJ) in plants. This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12-mediated MMEJ-based large-fragment deletion. The toolkit facilitates functional genomics and crop improvement, and the methodology is readily adaptable to other plant species.
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Employing CRISPR/Cas9 genome engineering to dissect the molecular requirements for mitosis.
The faithful execution of cell division requires the coordinated action of hundreds of gene products. Precisely perturbing these gene products in cells is central to understanding their functions during normal cell division, and the contributions of their disruption to disease. Here, we describe experimental approaches for using CRISPR/Cas9 for gene disruption and modification, with a focus on human cell culture. We describe strategies for inducible gene disruption to generate acute knockouts of essential cell division genes, which can be modified for the chronic elimination of nonessential genes. We also describe strategies for modifying the genome to generate protein fusions to report on and modify protein behavior. These tools facilitate investigation of protein function, dissection of protein assembly networks, and analyses of disease-associated mutations.
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A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii.
We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.
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Genotoxic effects of base and prime editing in human hematopoietic stem cells.
Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.
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Fine tuning wheat heading time through genome editing of transcription factor binding sites in Ppd-1 gene promoter.
Increasing the productivity and adaptability of agricultural plants often depends on optimizing heading time. To develop common wheat lines with accelerated heading and investigate its regulation, we targeted the PPD-1 genes, which control photoperiod sensitivity. Large deletions in the promoter regions of these genes are known to disrupt their expression, resulting in early heading. Using CRISPR/Cas9 genome editing, we generated wheat plants with mutations in the promoter regions of the Ppd-D1 and Ppd-B1 genes. These mutations included nucleotide substitutions, deletions, and insertions ranging from several to hundreds of base pairs, occurring within probable transcription factor binding sites, that may influence gene expression. Under short-day conditions, we assessed PPD-1 gene expression in T0 plants and T2 lines with different mutations. Our analysis revealed that deletions spanning the CHE transcription repressor binding sites altered gene expression patterns, supporting the hypothesis regarding the role of these cis-elements in regulating PPD-1 expression. Furthermore, plants with different mutations displayed distinct diurnal expression patterns, suggesting the involvement of additional transcription factors in the regulation of this gene. Evaluation of heading time in T1 and T2 families with different mutations demonstrated that plants with mutations affecting the "core region", including the CHE binding sites, initiated heading significantly earlier than those without mutations.
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Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.
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Cardiovascular Disease and Long Noncoding RNAs: Tools for Unraveling the Mystery Lnc-ing RNA and Phenotype.
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