Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants.

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared with the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice, significantly limiting their application. Here, we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice. We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID∗Δ, and TadA-CDd outperformed TadA-E27R/N46L. A C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P. In addition, a dual base editor constructed with a single protein, TadDE, enabled simultaneous, highly efficient C-to-T and A-to-G editing in rice. Collectively, our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice, providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

Boosting genome editing in plants with single transcript unit surrogate reporter systems.

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of editing events. In this study, we introduce multiple single transcript unit surrogate reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are used to restore functional reporter genes after genome editing, including efficient single-strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-base editor systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are shown to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using 'digital' genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Base editing and prime editing in laboratory animals.

Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple 'search and replace' editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.

CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Novel Wx alleles generated by base editing for improvement of rice grain quality.

Amylose content (AC), which is regulated by the Waxy (Wx) gene, is a major indicator of eating and cooking quality (ECQ) in rice (Oryza sativa). Thus far, only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein. Here, nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors. The nucleotide substitutions led to changes in 15 amino acid residues of Wx, and a series of novel Wx alleles with ACs of 0.3%-29.43% (wild type with AC of 19.87%) were obtained. Importantly, the waxyabe2 allele showed a "soft rice" AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxyabe2 progeny, generating a promising breeding material for improving rice grain quality.

Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences.

The most popular CRISPR-SpCas9 system recognizes canonical NGG protospacer adjacent motifs (PAMs). Previously engineered SpCas9 variants, such as Cas9-NG, favor G-rich PAMs in genome editing. In this manuscript, we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAMs. This study fills a major technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, which greatly expands the targeting scope of CRISPR-Cas9. Finally, our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems, facilitating easy adoption of the systems by others. We anticipate that more tools, such as prime editing, homology-directed repair, CRISPR interference, and CRISPR activation, will be further developed based on our promising iSpyMacCas9 platform.

The Scope of Pathogenic ABCA4 Mutations Targetable by CRISPR DNA Base Editing Systems-A Systematic Review.

Stargardt macular dystrophy (STGD1) is the most common form of inherited childhood blindness worldwide and for which no current treatments exist. It is an autosomal recessive disease caused by mutations in ABCA4. To date, a variety of gene supplementation approaches have been tested to create a therapy, with some reaching clinical trials. New technologies, such as CRISPR-Cas based editing systems, provide an exciting frontier for addressing genetic disease by allowing targeted DNA or RNA base editing of pathogenic mutations. ABCA4 has ∼1,200 known pathogenic mutations, of which ∼63% are transition mutations amenable to this editing technology. In this report, we screened the known "pathogenic" and "likely pathogenic" mutations in ABCA4 from available data in gnomAD, Leiden Open Variation Database (LOVD), and ClinVar for potential PAM sites of relevant base editors, including Streptococcus pyogenes Cas (SpCas), Staphylococcus aureus Cas (SaCas), and the KKH variant of SaCas (Sa-KKH). Overall, of the mutations screened, 53% (ClinVar), 71% (LOVD), and 71% (gnomAD), were editable, pathogenic transition mutations, of which 35-47% had "ideal" PAM sites. Of these mutations, 16-20% occur within a range of multiple PAM sites, enabling a variety of editing strategies. Further, in relevant patient data looking at three cohorts from Germany, Denmark, and China, we find that 44-76% of patients, depending on the presence of complex alleles, have at least one transition mutation with a nearby SaCas, SpCas, or Sa-KKH PAM site, which would allow for potential DNA base editing as a treatment strategy. Given the complexity of the genetic landscape of Stargardt, these findings provide a clearer understanding of the potential for DNA base editing approaches to be applied as ABCA4 gene therapy strategies.