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Base Editing: Efficient Installation of Point Mutations with Minimal Byproducts.
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Application of CRISPR-Cas9-Based Gene Editing Technology in Inherited Liver Diseases.
Inherited liver diseases are predominantly caused by monogenic mutations, and the vast majority of these conditions currently lack curative treatment options. Although liver transplantation may be used for patients with end-stage disease, it faces numerous challenges, including donor organ shortage, immune rejection, and the need for lifelong immunosuppression. In recent years, CRISPR-Cas9-based gene editing technology has advanced rapidly, offering transformative hope for the treatment of these diseases. This review systematically elucidates the working principles and technical advantages of the CRISPR-Cas9 system and its derived tools (base editing and prime editing), summarizes recent applications of these technologies in the treatment of hereditary liver diseases, and discusses the prospects and challenges of their clinical translation, aiming to provide a theoretical reference for future research in this field.
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In vivo somatic cell base editing and prime editing.
Recent advances in genome editing technologies have magnified the prospect of single-dose cures for many genetic diseases. For most genetic disorders, precise DNA correction is anticipated to best treat patients. To install desired DNA changes with high precision, our laboratory developed base editors (BEs), which can correct the four most common single-base substitutions, and prime editors, which can install any substitution, insertion, and/or deletion over a stretch of dozens of base pairs. Compared to nuclease-dependent editing approaches that involve double-strand DNA breaks (DSBs) and often result in a large percentage of uncontrolled editing outcomes, such as mixtures of insertions and deletions (indels), larger deletions, and chromosomal rearrangements, base editors and prime editors often offer greater efficiency with fewer byproducts in slowly dividing or non-dividing cells, such as those that make up most of the cells in adult animals. Both viral and non-viral in vivo delivery methods have now been used to deliver base editors and prime editors in animal models, establishing that base editors and prime editors can serve as effective agents for in vivo therapeutic genome editing in animals. This review summarizes examples of in vivo somatic cell (post-natal) base editing and prime editing and prospects for future development.
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Cytosine base editors (CBEs) for inducing targeted DNA base editing in Nicotiana benthamiana.
BACKGROUND: The base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration. RESULTS: In the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs containing A3A, A3A (Y130F), and rAPOBEC1(R33A) to compare their base editing efficiency in allotetraploid N. benthamiana (n = 4x). We used 14 target sites to compare their editing efficiency using transient transformation in tobacco plants. The sanger sequencing and deep sequencing results showed that A3A-CBE was the most efficient base editor. In addition, the results showed that A3A-CBE provided most comprehensive editing window (C1 ~ C17 could be edited) and had a better editing efficiency under the base background of TC. The target sites (T2 and T6) analysis in transformed N. benthamiana showed that only A3A-CBE can have C-to-T editing events and the editing efficiency of T2 was higher than T6. Additionally, no off-target events were found in transformed N. benthamiana. CONCLUSIONS: All in all, we conclude that A3A-CBE is the most suitable vector for specific C to T conversion in N. benthamiana. Current findings will provide valuable insights into selecting an appropriate base editor for breeding polyploid plants.
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Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA.
Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5' end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing.IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.
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Advancing gene-editing platforms to improve the viability of rare-disease therapeutics: key insights from a 2024 Scientific Exchange hosted by ARM, ISCT, and Danaher.
Rare-disease therapeutics face viability challenges due to small patient populations and drug-development and regulatory frameworks that were not developed to address rapidly progressive or quickly fatal conditions. Because the majority of rare diseases are genetic in nature, gene-editing modalities offer substantial promise. This Scientific Exchange, co-hosted by the Alliance for Regenerative Medicine, the International Society for Cell and Gene Therapy, and Danaher Corporation in November 2024, set out to address the challenge of realizing the full promise of gene editing for rare-disease therapies by advancing platforms that leverage stable and reusable processes or components to develop multiple therapies. Through multi-stakeholder engagement and discussions of case studies in CRISPR/Cas nuclease, base, and prime editing, 4 key opportunities emerged that deliver value by holding platform elements constant and/or streamlining development steps: (1) consistent delivery vehicle; (2) consistent manufacturing; (3) benefit-risk appropriate quality requirements; and (4) expansive clinical trial designs. Together, these opportunities could yield up to 5-fold efficiency gains and result in substantial value creation for patients, regulators, and developers, potentially decreasing the time required to dose patients with a new gene-editing therapy from years down to 6 months.
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Pioneer Factor Improves CRISPR-Based C-To-G and C-To-T Base Editing.
Base editing events in eukaryote require a compatible chromatin environment, but there is little research on how chromatin factors contribute to the editing efficiency or window. By engineering BEs (base editors) fused with various pioneer factors, the authors found that SOX2 substantially increased the editing efficiency for GBE and CBE. While SoxN-GBE (SOX2-NH3-GBE) improved the editing efficiency at overall cytosines of the protospacer, SoxM-GBE/CBE (SOX2-Middle-GBE/CBE) enabled the higher base editing at PAM-proximal cytosines. By separating functional domains of SOX2, the SadN-GBE (SOX2 activation domain-NH3-GBE) is constructed for higher editing efficiency and SadM-CBE for broader editing window to date. With the DNase I assay, it is also proved the increased editing efficiency is most likely associated with the induction of chromatin accessibility by SAD. Finally, SadM-CBE is employed to introduce a stop codon in the proto-oncogene MYC, at a locus rarely edited by previous editors with high efficiency. In this work, a new class of pioneer-BEs is constructed by fusion of pioneer factor or its functional domains, which exhibits higher editing efficiency or broader editing window in eukaryote.
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The "new favorite" of gene editing technology-single base editors.
Programmable nucleases are cutting edge genetic technology which edits targeted DNA sequences through generation of site-specific double-strand DNA breaks (DSBs). To improve the efficiency and precision of genetic modification, scientists have developed a single-base editing system (base editor) through combining of CRISPR/Cas9 system with cytosine deaminase. Compared with Cas9 system, this base editor can convert cytosine to thymine (C > T) at specific site more efficiently without inducing DSBs to avoid generation of indels. However, the base editor can only generate transition of pyrimidine but could not modify purines. Recently, Nature published a novel base editing system to convert adenine to guanine (ABEs, adenine base editors) through fusion of Cas9 nickase to a modified deaminase which is evolved through screening of random library based on tRNA adenine deaminase from E. coli. Here, we summarize the development of single-base editing tools and the latest research progress, especially the optimization process of ABEs, as well as the potential directions of the base editors.
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CRISPR/Cas9-Based Genome Editing Toolbox for Arabidopsis thaliana.
CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system. Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing in other plant species such as rice.
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Base editing: precision chemistry on the genome and transcriptome of living cells.
RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products. In this Review, we summarize base-editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision and in vivo delivery of base editors and discuss limitations and future directions of base editing for research and therapeutic applications.
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