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Diversification of plant agronomic traits by genome editing of brassinosteroid signaling family genes in rice.
Brassinosteroids (BRs) regulate various agronomic traits such as plant height, leaf angle, and grain size in rice (Oryza sativa L.); thus, BR signaling components are promising targets for molecular rational design. However, genetic materials for BR-signaling genes or family members remain limited in rice. Here, by genome editing using clustered regularly interspaced short palindromic repeats (CRSPR)/Cas9 tools, we generated a panel of single, double, triple, or quadruple mutants within three BR signaling gene families, including GSK3/SHAGGY-LIKE KINASE1 (GSK1)-GSK4, BRASSINAZOLE-RESISTANT1 (OsBZR1)-OsBZR4, and protein phosphatases with kelch-like (PPKL)1-PPKL3, under the same background (Zhonghua11, japonica). The high-order mutants were produced by either simultaneously targeting multiple sites on different genes of one family (GSKs and PPKLs) or targeting the overlapping sequences of family members (OsBZRs). The mutants exhibited a diversity of plant height, leaf angle, and grain morphology. Comparison analysis of the phenotypes together with BR sensitivity tests suggested the existence of functional redundancy, differentiation, or dominancy among the members within each family. In addition, we generated a set of transgenic plants overexpressing GSK2, OsBZR1/2, and PPKL2, respectively, in wild-type or activated forms with fusion of different tags, and also verified the protein response to BR application. Collectively, these plants greatly enriched the diversity of important agronomic traits in rice. We propose that editing of BR-related family genes could be a feasible approach for screening of desired plants to meet different requirements. Release of these materials as well as the related information also provides valuable resources for further BR research and utilization.
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Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants.
The Cas12j-8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5'-TTN-3' protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j-8 nuclease. The engineered crRNA and Cas12j-8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j-2 variant, nCas12j-2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j-8, demonstrating an average increase of 5.36- to 6.85-fold in base-editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j-8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.
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Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli.
Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.
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PAM-Less CRISPR-SpRY Genome Editing in Plants.
Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.
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Base editing of organellar DNA with programmable deaminases.
Mitochondria and chloroplasts are organelles that include their own genomes, which encode key genes for ATP production and carbon dioxide fixation, respectively. Mutations in mitochondrial DNA can cause diverse genetic disorders and are also linked to ageing and age-related diseases, including cancer. Targeted editing of organellar DNA should be useful for studying organellar genes and developing novel therapeutics, but it has been hindered by lack of efficient tools in living cells. Recently, CRISPR-free, protein-only base editors, such as double-stranded DNA deaminase toxin A-derived cytosine base editors (DdCBEs) and adenine base editors (ABEs), have been developed, which enable targeted organellar DNA editing in human cell lines, animals and plants. In this Review, we present programmable deaminases developed for base editing of organellar DNA in vitro and discuss mitochondrial DNA editing in animals, and plastid genome (plastome) editing in plants. We also discuss precision and efficiency limitations of these tools and propose improvements for therapeutic, agricultural and environmental applications.
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Simultaneous high-efficiency base editing and reprogramming of patient fibroblasts.
Human induced pluripotent stem cells (hiPSCs) allow in vitro study of genetic diseases and hold potential for personalized stem cell therapy. Gene editing, precisely modifying specifically targeted loci, represents a valuable tool for different hiPSC applications. This is especially useful in monogenic diseases to dissect the function of unknown mutations or to create genetically corrected, patient-derived hiPSCs. Here we describe a highly efficient method for simultaneous base editing and reprogramming of fibroblasts employing a CRISPR-Cas9 adenine base editor. As a proof of concept, we apply this approach to generate gene-edited hiPSCs from skin biopsies of four patients carrying a Finnish-founder pathogenic point mutation in either NOTCH3 or LDLR genes. We also show LDLR activity restoration after the gene correction. Overall, this method yields tens of gene-edited hiPSC monoclonal lines with unprecedented efficiency and robustness while considerably reducing the cell culture time and thus the risk for in vitro alterations.
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Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor.
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal for targeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities.
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Optimized gGBE enables robust G-to-Y conversion in mammalian cells and zebrafish embryos.
Base editing is a powerful and precise genome-editing tool capable of introducing single-base substitutions with high resolution. Depending on the base-modification domains employed, base editors can mediate both transition and transversion base editing in mammalian cells. While deaminase-derived base editors have enabled efficient transition editing in the zebrafish genome, glycosidase-derived transversion editing remains a significant challenge. In this study, guided by structural insights into the Cas9 complex, we engineered a series of glycosidase guanine base editor (gGBE) constructs by inlaying the N-methylpurine DNA glycosylase (MPG) domain into Cas9 protein. Among these variants, we identified a high-performance gGBE in which MPG was inserted between residues G1246 and S1247 of the PI domain in Streptococcus pyogenes Cas9, which we named gGBE-PIGS. This variant improved editing efficiency by up to 3.5-fold in mammalian cells and 13-fold in zebrafish embryos, without compromising editing precision. Furthermore, we developed a gGBE-editing-responsive GFP reporter to monitor editing efficiency and enrich for gGBE-edited cells and embryos. Using a combination of the optimized gGBE-PIGS and the GFP reporter, we successfully generated zebrafish models carrying mutations homologous to human amyotrophic lateral sclerosis (ALS)-associated variants. Together, these findings highlight the robustness and versatility of the gGBE platform for precise base editing and underscore its promising potential for in vivo disease modeling and therapeutic genome engineering.
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Precision genome editing strategies for enduring lipid lowering in atherosclerosis.
Atherosclerosis continues to be a primary contributor to global cardiovascular mortality, influenced by intricate lipid and inflammatory mechanisms. Despite the efficacy of conventional pharmacotherapies, ongoing issues of patient non-adherence and residual risk have prompted the exploration of more enduring therapeutic alternatives. This review examines the significant transition in cardiovascular research from conventional, wide knockout models to the utilization of advanced precision genome editing methods, particularly emphasizing CRISPR-Cas9, base editing, and prime editing. These sophisticated molecular tools allow for the accurate insertion and rectification of single-nucleotide variants without causing double-strand breaks, marking a significant shift from rudimentary gene disruption to precise variant engineering. By specifically targeting essential lipid-regulating genes like proprotein convertase subtilisin/kexin type 9 (PCSK9) and angiopoietin-like 3 (ANGPTL3), precision editing presents an exceptional opportunity for lasting, one-shot lipid-lowering treatments. Additionally, we examine the advancement of preclinical modeling, emphasizing humanized models that precisely represent population genetics. This review highlights the essential obstacles to clinical translation, focusing on the optimization of delivery systems such as adeno-associated viruses (AAVs) and lipid nanoparticles (LNPs), as well as the thorough assessment of off-target effects and ethical implications.
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Towards therapeutic base editing.
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