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A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants.
Genetic mutations in BRCA1, which is crucial for the process of DNA repair and maintenance of genomic integrity, are known to increase markedly the risk of breast and ovarian cancers. Clinical genetic testing has been used to identify new BRCA1 variants; however, functional assessment and determination of their pathogenicity still poses challenges for clinical management. Here, we describe that CRISPR-mediated cytosine base editor, known as BE3, can be used for the functional analysis of BRCA1 variants. We performed CRISPR-mediated base-editing screening using 745 gRNAs targeting all exons in BRCA1 to identify loss-of-function variants and identified variants whose function has heretofore remained unknown, such as c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A. Our results show that CRISPR-mediated base editor is a powerful tool for the reclassification of variants of uncertain significance (VUSs) in BRCA1.
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Gene editing for collagen disorders: current advances and future perspectives.
Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from localised tissue abnormalities to severe systemic complications. Symptoms differ significantly and depend on the pathogenic variant, which can affect various systems, including the musculoskeletal, cardiovascular, and respiratory systems, highlighting the complex implications of collagen gene pathogenic variants and the wide range of expression patterns among different collagen types. Gene-editing technologies, particularly Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas systems, have emerged as promising therapeutic options for these disorders, representing a putative one-for-all treatment strategy. This review provides an overview of current gene-editing strategies aimed at collagen-related diseases, including osteogenesis imperfecta, Alport syndrome, and dystrophic epidermolysis bullosa. We explore the application of CRISPR-Cas9, which facilitates targeted DNA modifications, base editing (BE), and prime editing (PE), enabling precise single-nucleotide alterations without double-strand breaks (DSB). Preclinical and clinical studies have shown the potential of gene therapy to enhance collagen production, restore tissue integrity, and alleviate symptoms. However, challenges persist, including the lack of recurring mutations, the need for improved delivery methods, the reduction of off-target effects, and the development of novel therapies. Despite these challenges, advancements in gene editing techniques appear promising in enhancing editing efficiency while minimising unintended mutations, paving the way for more precise and safer genetic interventions for collagen disorders. Gene editing is fundamentally transforming medicine and biotechnology. Its applications encompass advanced diagnostics, tailored therapeutic strategies, and solutions for rare genetic disorders. By enabling precise genetic modifications, gene editing is paving the way for treatments of previously untreatable diseases, including those linked to collagen pathogenic variants. This review discusses the latest advancements in gene therapy techniques targeting collagen-related disorders. It explores innovative approaches like CRISPR-Cas9-mediated gene editing and highlights emerging strategies, such as allele-specific inactivation and base editing (BE). By examining these cutting-edge therapies and their potential clinical applications, this review highlights the transformative impact of gene editing in treating collagen-related conditions, while also identifying critical challenges and future directions for research.
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Ligase-mediated programmable genomic integration (L-PGI).
Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.
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Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean.
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean (Glycinemax). The Streptococcuspyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5'-NGG-3' (SpCas9) or 5'-TTTN-3' (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5'-NNGRRT-3' (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5'-AAGGGT-3', 5'-GGGGAT-3', 5'-TTGAAT-3', and 5'-TAGGGT-3' in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.
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CRISPR-Cas applications in agriculture and plant research.
Growing world population and deteriorating climate conditions necessitate the development of new crops with high yields and resilience. CRISPR-Cas-mediated genome engineering presents unparalleled opportunities to engineer crop varieties cheaper, easier and faster than ever. In this Review, we discuss how the CRISPR-Cas toolbox has rapidly expanded from Cas9 and Cas12 to include different Cas orthologues and engineered variants. We present various CRISPR-Cas-based methods, including base editing and prime editing, which are used for precise genome, epigenome and transcriptome engineering, and methods used to deliver the genome editors into plants, such as bacterial-mediated and viral-mediated transformation. We then discuss how promoter editing and chromosome engineering are used in crop breeding for trait engineering and fixation, and important applications of CRISPR-Cas in crop improvement, such as de novo domestication and enhancing tolerance to abiotic stresses. We conclude with discussing future prospects of plant genome engineering.
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CRISPR/Cas Genome Editing Technologies for Plant Improvement against Biotic and Abiotic Stresses: Advances, Limitations, and Future Perspectives.
Crossbreeding, mutation breeding, and traditional transgenic breeding take much time to improve desirable characters/traits. CRISPR/Cas-mediated genome editing (GE) is a game-changing tool that can create variation in desired traits, such as biotic and abiotic resistance, increase quality and yield in less time with easy applications, high efficiency, and low cost in producing the targeted edits for rapid improvement of crop plants. Plant pathogens and the severe environment cause considerable crop losses worldwide. GE approaches have emerged and opened new doors for breeding multiple-resistance crop varieties. Here, we have summarized recent advances in CRISPR/Cas-mediated GE for resistance against biotic and abiotic stresses in a crop molecular breeding program that includes the modification and improvement of genes response to biotic stresses induced by fungus, virus, and bacterial pathogens. We also discussed in depth the application of CRISPR/Cas for abiotic stresses (herbicide, drought, heat, and cold) in plants. In addition, we discussed the limitations and future challenges faced by breeders using GE tools for crop improvement and suggested directions for future improvements in GE for agricultural applications, providing novel ideas to create super cultivars with broad resistance to biotic and abiotic stress.
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ABE-ultramax for high-efficiency biallelic adenine base editing in zebrafish.
Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, base editors significantly accelerate and refine in vivo analysis of genetic variations. However, base editors are restricted by protospacer adjacent motif (PAM) sequences and specific editing windows, hindering their applicability to a broad spectrum of genetic variants. Additionally, base editors can introduce unintended mutations and often exhibit reduced efficiency in living organisms compared to cultured cell lines. Here, we engineer a suite of adenine base editors (ABEs) called ABE-Ultramax (Umax), demonstrating high editing efficiency and low rates of insertions and deletions (indels) in zebrafish. The ABE-Umax suite of editors includes ABEs with shifted, narrowed, or broadened editing windows, reduced bystander mutation frequency, and highly flexible PAM sequence requirements. These advancements have the potential to address previous challenges in disease modeling and advance gene therapy applications.
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Engineering Agrobacterium for improved plant transformation.
Outside of a few model systems and selected taxa, the insertion of transgenes and regeneration of modified plants are difficult or impossible. This is a major bottleneck both for biotechnology and scientific research with many important species. Agrobacterium-mediated transformation (AMT) remains the most common approach to insert DNA into plant cells, and is also an important means to stimulate regeneration of organized tissues. However, the strains and transformation methods available today have been largely unchanged since the 1990s. New sources of Agrobacterium germplasm and associated genomic information are available for hundreds of wild strains in public repositories, providing new opportunities for research. Many of these strains contain novel gene variants or arrangements of genes in their T-DNA, potentially providing new tools for strain enhancement. There are also several new techniques for Agrobacterium modification, including base editing, CRISPR-associated transposases, and tailored recombineering, that make the process of domesticating wild strains more precise and efficient. We review the novel germplasm, genomic resources, and new methods available, which together should lead to a renaissance in Agrobacterium research and the generation of many new domesticated strains capable of promoting plant transformation and/or regeneration in diverse plant species.
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BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors.
Adenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.
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Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.
CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.
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