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Identification of functional sgRNA mutants lacking canonical secondary structure using high-throughput FACS screening.
Coexpressing multiple identical single guide RNAs (sgRNAs) in CRISPR-dependent engineering triggers genetic instability and phenotype loss. To provide sgRNA derivatives for efficient DNA digestion, we design a high-throughput digestion-activity-dependent positive screening strategy and astonishingly obtain functional nonrepetitive sgRNA mutants with up to 48 out of the 61 nucleotides mutated, and these nonrepetitive mutants completely lose canonical secondary sgRNA structure in simulation. Cas9-sgRNA complexes containing these noncanonical sgRNAs maintain wild-type level of digestion activities in vivo, indicating that the Cas9 protein is compatible with or is able to adjust the secondary structure of sgRNAs. Using these noncanonical sgRNAs, we achieve multiplex genetic engineering for gene knockout and base editing in microbial cell factories. Libraries of strains with rewired metabolism are constructed, and overproducers of isobutanol or 1,3-propanediol are identified by biosensor-based fluorescence-activated cell sorting (FACS). This work sheds light on the remarkable flexibility of the secondary structure of functional sgRNA.
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Molecular mechanisms and biotechnological advances in herbicide resistance: Insights into the development of herbicide-tolerant crops.
Herbicides play a pivotal role in modern agriculture by controlling weed populations and safeguarding crop yields. However, the long-term and extensive use of herbicides has accelerated the evolution of herbicide-resistant weeds, thereby diminishing their efficacy and posing a serious threat to global food security. Recent advances in molecular biology and plant biotechnology have greatly expanded our understanding of herbicide resistance mechanisms and enabled the development of crops with enhanced herbicide resistance. Herbicide resistance genes function primarily by encoding detoxifying enzymes, modifying herbicide target sites, or activating specific metabolic pathways that mitigate herbicidal toxicity. Emerging genetic tools, including transgenic approaches and CRISPR/Cas-mediated genome editing, have further facilitated the precise introduction of resistance traits into major crops. It is noteworthy that this review offers novel insights into the latest CRISPR/Cas applications, including base editing and prime editing for developing novel, non-transgenic herbicide-resistant crops. Furthermore, it provides a systematic overview of advanced strategies for engineering multi-gene stacking traits to combat complex or evolving weed resistance. This review integrates recent progress in elucidating the molecular targets of herbicides and the underlying resistance mechanisms, and highlights the potential of modern biotechnological strategies for engineering herbicide-resistant crops to promote sustainable and environmentally responsible weed management.
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Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases.
Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.
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Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems.
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene-editing tools. As the natural inhibitors of CRISPR-Cas systems, anti-CRISPRs (Acrs) can be used as the "off-switch" for CRISPR-Cas systems to limit the off-target effects caused by Cas9. Since the discovery of CRISPR-Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.
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Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair.
The ability to alter single bases without homology directed repair (HDR) of double-strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5, which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor (BE3) plasmid and four different sgRNAs into caprine fibroblasts were ineffective in altering FGF5. In contrast, all five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF5 expression. PCR amplicon and whole-genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single-cell embryos. Our results support further optimization of BEs for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation.
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Prime editing for functional repair in patient-derived disease models.
Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.
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CRISPR-based gene editing technology and its application in microbial engineering.
Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated technologies have provided an efficient tool for genetic engineering of cells and organisms. Here, we review the three emerging gene editing tools (ZFNs, TALENs, and CRISPR-Cas) and briefly introduce the principle, classification, and mechanisms of the CRISPR-Cas systems. Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems, as well as the novel base editor (BE), prime editor (PE), and CRISPR-associated transposase (CAST) technologies, are described in detail. In addition, we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications. Finally, the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.
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Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.
Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.
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CRISPR-mediated ablation of TP53 and EGFR mutations enhances gefitinib sensitivity and anti-tumor efficacy in lung cancer.
Multiple pathogenic single-nucleotide polymorphisms (SNPs) have been identified as contributing factors in the aggravation of cancer prognosis and emergence of drug resistance in various cancers. Here, we targeted mutated EGFR and TP53 oncogenes harboring single-nucleotide missense mutations (EGFR-T790M and TP53-R273H) that are associated with gefitinib resistance. Co-delivery of adenine base editor (ABE) and EGFR- and TP53-SNP specific single-guide RNA via adenovirus (Ad) resulted in precise correction of the oncogenic mutations with high accuracy and efficiency in vitro and in vivo. Importantly, compared with a control group treated only with gefitinib, an EGFR inhibitor, co-treatment with Ad/ABE targeting SNPs in TP53 and EGFR in combination with gefitinib increased drug sensitivity and suppressed abnormal tumor growth more efficiently. Taken together, these results indicate that ABE-mediated correction of dual oncogenic SNPs can be an effective strategy for the treatment of drug-resistant cancers.
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Engineering Oncogenic Hotspot Mutations on SF3B1 via CRISPR-Directed PRECIS Mutagenesis.
UNLABELLED: SF3B1 is the most recurrently mutated RNA splicing gene in cancer. However, research of its pathogenic role has been hindered by a lack of disease-relevant cell line models. Here, our study compared four genome engineering platforms to establish SF3B1 mutant cell lines: CRISPR-Cas9 editing, AAV homology-directed repair editing, base editing (ABEmax, ABE8e), and prime editing (PE2, PE3, PE5max). We showed that prime editing via PE5max achieved the most efficient SF3B1 K700E editing across a wide range of cell lines. Our approach was further refined by coupling prime editing with a fluorescent reporter that leverages a SF3B1 mutation-responsive synthetic intron to mark successfully edited cells. By applying this approach, called prime editing coupled intron-assisted selection (PRECIS), we introduced the K700E hotspot mutation into two chronic lymphocytic leukemia cell lines, HG-3 and MEC-1. We demonstrated that our PRECIS-engineered cells faithfully recapitulate known mutant SF3B1 phenotypes, including altered splicing, copy number variations, and cell-growth defect. Moreover, we discovered that the SF3B1 mutation can cause the loss of Y chromosome in chronic lymphocytic leukemia. Our results showcase that PRECIS is an efficient and generalizable method for engineering genetically faithful SF3B1 mutant models. Our approach provides new insights on the role of SF3B1 mutation in cancer and enables the generation of SF3B1 mutant cell lines in relevant cellular context. SIGNIFICANCE: This study developed an approach that can reliably and efficiently engineer SF3B1 mutation into different cellular contexts, thereby revealing novel roles of SF3B1 mutation in driving aberrant splicing, clonal evolution, and genome instability.
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