Cytidine Base Editors Research Articles

CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing system has altered plant research by allowing for targeted genome alteration, and they are emerging as powerful tools for evaluating plant gene function and improving crop yield. Even though CRISPR/Cas9 cleavage and subsequent repair are effective ways to precisely replace genes and change base pairs in plants, the dominance of the non-homologous end-joining pathway (NHEJ) and homology-directed repair's (HDR) poor effectiveness in plant cells have restricted their use. Base editing is gaining popularity as a potential alternative to HDR or NHEJ-mediated replacement, allowing for precise changes in the plant genome via programmed conversion of a single base to another without the need for a donor repair template or double-stranded breaks. In this review, we primarily present the mechanisms of base-editing system, including their distinct types such as DNA base editors (cytidine base editor and adenine base editor) and RNA base editors discovered so far. Next, we outline the current potential applications of the base-editing system for crop improvements. Finally, we discuss the limitations and potential future directions of the base-editing system in terms of improving crop quality. We hope that this review will enable the researcher to gain knowledge about base-editing tools and their potential applications in crop improvement.

Accelerated discovery and miniaturization of novel single-stranded cytidine deaminases.

Cytidine base editors (CBEs) hold significant potential in genetic disease treatment and in breeding superior traits into animals. However, their large protein sizes limit their delivery by adeno-associated virus (AAV), given its packing capacity of <4.7 kb. To overcome this, we employed a web-based fast generic discovery (WFG) strategy, identifying several small ssDNA deaminases (Sdds) and constructing multiple Sdd-CBE 1.0 versions. SflSdd-CBE 1.0 demonstrated high C-to-T editing efficiency, comparable to AncBE4max, while SviSdd-CBE 1.0 exhibited moderate C-to-T editing efficiency with a narrow editing window (C3 to C5). Utilizing AlphaFold2, we devised a one-step miniaturization strategy, reducing the size of Sdds while preserving their efficiency. Notably, we administered AAV8 expressing PCSK9 targeted sgRNA and SflSdd-CBEs (nSaCas9) 2.0 into mice, leading to gene-editing events (with editing efficiency up to 15%) and reduced serum cholesterol levels, underscoring the potential of Sdds in gene therapy. These findings offer new single-stranded editing tools for the treatment of rare genetic diseases.

Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing

Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria.

A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.

Highly efficient in vivo C-to-T base editing in Atlantic salmon (Salmo salar) – A step towards aquaculture precision breeding

The salmon industry has the potential to provide nutritious food to the expanding world population. However, the industry faces several sustainability challenges related to environmental impact and fish welfare. Genome editing presents a promising approach for precision breeding, where small, targeted changes to the fish genome may facilitate the introduction of advantageous traits like disease resistance, delayed maturation, or enhanced growth. This study presents the first application of base editing in an aquaculture fish species. By co-injecting cytidine base editor AncBE4max mRNA and gRNA targeting the pigmentation gene solute carrier family 45 member 2 in Atlantic salmon embryos, we were able to induce a specific C-to-T conversion, thereby introducing a premature stop codon and achieving an albino phenotype. High-throughput sequencing revealed an average of 50–66% correct conversion of the target base depending on the base editor mRNA concentration and up to 89% in a single individual. Our results suggest that AncBE4max provides a simple and efficient approach to making precise single nucleotide edits in the Atlantic salmon genome. Additionally, we used conventional CRISPR/Cas9 combined with oligonucleotide templates to insert point mutations at various positions up- and downstream of the Cas9 cleavage site, demonstrating that the insertion efficiency was affected by the position of the mutation. The application of genome editing tools that allow for precise changes in this species is a step towards aquaculture precision breeding and the introduction of traits promoting a more sustainable industry.

Efficient exon skipping by base-editor-mediated abrogation of exonic splicing enhancers

Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.

CRISPR/Cas9-based Genome Editing of Pseudomonas aeruginosa.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been developed as a robust genome engineering tool in a variety of organisms attributed to its high efficiency and versatility. In this chapter, we described the detailed procedures of CRISPR-Cas9-based genetic manipulation in Pseudomonas aeruginosa, including precise gene deletion and insertion via Cas9-mediated DNA double-strand break and homologous recombination repair. In addition, we provided a detailed protocol for cytidine base editor, a highly efficient gene inactivation and point mutation tool in Pseudomonas aeruginosa.

Application of CRISPR-Cas System to Mitigate Superbug Infections.

Multidrug resistance in bacterial strains known as superbugs is estimated to cause fatal infections worldwide. Migration and urbanization have resulted in overcrowding and inadequate sanitation, contributing to a high risk of superbug infections within and between different communities. The CRISPR-Cas system, mainly type II, has been projected as a robust tool to precisely edit drug-resistant bacterial genomes to combat antibiotic-resistant bacterial strains effectively. To entirely opt for its potential, advanced development in the CRISPR-Cas system is needed to reduce toxicity and promote efficacy in gene-editing applications. This might involve base-editing techniques used to produce point mutations. These methods employ designed Cas9 variations, such as the adenine base editor (ABE) and the cytidine base editor (CBE), to directly edit single base pairs without causing DSBs. The CBE and ABE could change a target base pair into a different one (for example, G-C to A-T or C-G to A-T). In this review, we addressed the limitations of the CRISPR/Cas system and explored strategies for circumventing these limitations by applying diverse base-editing techniques. Furthermore, we also discussed recent research showcasing the ability of base editors to eliminate drug-resistant microbes.

Cytosine base editors optimized for genome editing in potato protoplasts.

In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3-4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.

Development of PmCDA1-based high-efficiency cytidine base editors (ChyCBEs) incorporating a GmRad51 DNA-binding domain in soybean

Although many efficient base editing tools have been developed, there is still a need for more efficient CBEs in many important crop species. For instance, the BE editing efficiency in soybean still falls behind other model crops. In this study, we first generated an improved CBE configuration, using two copy UGI and a single transcript unit, and found that PmCDA1 is more effective than other tested base editors in soybean. We next developed a novel PmCDA1-based hyperdrive CBE (ChyBE03) by introducing a newly designed linker using a soybean GmRad51 ssDBD domain. We next used ChyBE03 to install a predefined stop codon in the Nod factor receptor gene (NFR1a). ChyBE03 showed up to 52.6% efficiency and can generate homozygous nfr1a mutants with desired non-nodulation phenotype in stable transgenic soybean. We also developed a ChyBE-NG with an expanded targeting scope and high editing efficiency. In conclusion, we developed a new PmCDA1-based high efficiency CBE tool, which is highly efficient and can be expanded for relaxed targeting scope with the SpCas9-NG variants.

CRISPR single base-editing: in silico predictions to variant clonal cell lines.

Engineering single base edits using CRISPR technology including specific deaminases and single-guide RNA (sgRNA) is a rapidly evolving field. Different types of base edits can be constructed, with cytidine base editors (CBEs) facilitating transition of C-to-T variants, adenine base editors (ABEs) enabling transition of A-to-G variants, C-to-G transversion base editors (CGBEs) and recently adenine transversion editors (AYBE) that create A-to-C and A-to-T variants. The base-editing machine learning algorithm BE-Hive predicts which sgRNA and base editor combinations have the strongest likelihood of achieving desired base edits. We have used BE-Hive and TP53 mutation data from The Cancer Genome Atlas (TCGA) ovarian cancer cohort to predict which mutations can be engineered, or reverted to wild-type (WT) sequence, using CBEs, ABEs or CGBEs. We have developed and automated a ranking system to assist in selecting optimally designed sgRNA that considers the presence of a suitable protospacer adjacent motif (PAM), the frequency of predicted bystander edits, editing efficiency and target base change. We have generated single constructs containing ABE or CBE editing machinery, an sgRNA cloning backbone and an enhanced green fluorescent protein tag (EGFP), removing the need for co-transfection of multiple plasmids. We have tested our ranking system and new plasmid constructs to engineer the p53 mutants Y220C, R282W and R248Q into WT p53 cells and shown that these mutants cannot activate four p53 target genes, mimicking the behaviour of endogenous p53 mutations. This field will continue to rapidly progress, requiring new strategies such as we propose to ensure desired base-editing outcomes.

PAM-flexible Cas9-mediated base editing of a hemophilia B mutation in induced pluripotent stem cells

BackgroundBase editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility.MethodsWe generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient’s F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively.ResultsHere we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG–mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor.ConclusionA base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.

A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.

CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.

Base editing screens map mutations affecting interferon-γ signaling in cancer.

Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive Tcells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.

A/C Simultaneous Conversion Using the Dual Base Editor in Human Cells.

Base editors, mostly cytidine base editors (CBEs) and adenine base editors (ABEs), are powerful tools for precise base editing. However, current base editors can only edit either adenines or cytosines. Thus, our lab has developed a dual base editor (A&C-BEmax) through the fusion of cytidine and adenosine deaminases to Cas9n to achieve both C•G to T•A and A•T to G•C mutations, which enables A/C simultaneous conversion in the same allele (up to 30%) in human cells. Here, we described a protocol for the usage of A&C-BEmax in human cells. This protocol includes standard dual base editing experiments in HEK293T cells and data analysis of dual base editing outcomes using BE-analyzer. All the workflow of experiments can be completed within 2-3weeks.

Targeted Base Editing Strategies for Beta-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks. In this study, we have developed BE strategies aiming either to correct the genetic defect or to target disease modifiers in patient cells, and provide a safe and effective treatment for β-hemoglobinopathies. First, we exploited the capacity of ABEs in combination with single guide RNAs (gRNAs) to correct prevalent β-thalassemic mutations (the CD39 and IVS2-1 β0 mutations and the IVS1-110 β+ mutation). RBCs derived from edited HSPCs exhibited high β-globin levels. The delayed erythroid differentiation typically observed in β-thalassemic cell cultures was corrected by our treatment. Measurement of Annexin+ cells by flow cytometry showed a substantially reduced apoptotic rate in edited β-thalassemic samples compared to untreated controls. Finally, off-target activity was minimal and by-stander edits (additional base conversions occurring close to the target base) did not impact the β-globin expression. In parallel, we used BE to target disease modifiers such as the fetal γ-globin genes. In fact, the co-inheritance of genetic mutations causing a sustained fetal β-like γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin (HbF), HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 eitherdisrupt the binding sites (BS) for transcriptional repressors (e.g., LRF) or generate BS for transcriptional activators (e.g., KLF1). BE allowed the introduction of HPFH mutations in the HBG promoters, which disrupt the LRF BS (via CBE) or generate the KLF1 BS (via ABE). This led to therapeutically relevant HbF levels in RBCs differentiated from edited β-thalassemia or SCD HSPCs, and rescued the pathological phenotypes (correcting ineffective erythropoiesis in β-thalassemia and the sickling phenotype in SCD RBCs). Finally, serial xenotransplantation experiments showed BE in long-term HSCs; however, the efficiency was reduced compared to input HSPCs. RNA-seq analysis showed no alteration for ABE-treated HSPCs, but activation of the p53 pathway and dysregulation of genes involved in HSC biology in CBE-treated samples. To increase the fitness of edited HSCs and editing efficiency, we optimized the RNA delivery of the BE machinery and used histone deacetylase, RNase and apoptosis inhibitors. Moreover, we developed a strategy to enrich edited cells. By way of example, two rounds of mRNA electroporation led to increased BE efficiency with minimal toxicity. Furthermore, highly processive base editors outperformed the original enzymes even upon only one round of electroporation. In conclusion, we developed efficient BE approaches to either correct highly prevalent mutations or induce HbF expression. Overall, our treatments led to correction of the pathological phenotypes and optimal BE efficiencies to finally enable the clinical development of base-edited HSPCs for β-hemoglobinopathies.

Two Is Better Than One: Fetal Hemoglobin Reactivation and Alpha-Globin Downregulation to Correct the β-Hemoglobinopathy Phenotype through Base Editing

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks, thus allowing the multiplexed editing of several targets. Mutations leading to elevated fetal Hb (HbF) or reduced α-globin expression are associated with a better clinical phenotype in both β-thalassemia and SCD patients. Fetal β-like γ-globin compensates for adult β-globin deficiency and reduced α-globin levels impairs its precipitation and inhibit Hb polymerization in SCD. In this study, we have developed BE strategies aiming to (i) reactivate HbF by down-regulating BCL11A, a master repressor of γ-globin expression though inactivation of its enhancers, and (ii) reduce the α-globin levels through targeting of the MCS-R2 enhancer of the α-globin genes (HBA1/2). First, we used CBEs and ABEs to target the GATA1 or ATF4 activator binding site (BS) within the erythroid-specific BCL11A enhancers (+58-kb and +55-kb regions) in SCD HSPCs through RNA delivery and achieved efficiencies of up to ~90%. A colony forming cell (CFC) assay showed no impact on progenitors' viability. RBCs derived from base-edited HSPCs exhibited high HbF expression levels that were sufficient to ameliorate the sickling phenotype. Samples carrying different mutations within the GATA1 or ATF4 BS were associated with different HbF levels, implying that some nucleotides are more essential for GATA1 and ATF4 binding and the consequent BCL11A gene activation. In parallel, we used CBEs and ABEs to target the GATA1 or NF-E2 activator BS within the MCS-R2 enhancer of HBA1/2. We screened single guide RNAs (sgRNAs) in an erythroid cell line (K562) that allowed editing of different nucleotides within the GATA1 and NF-E2 BS and we identified the most efficient sgRNAs diminishing α-globin expression. Next, selected sgRNAs were delivered to SCD HSPCs, achieving high editing efficiency (~85%). A CFC assay showed a good progenitor viability. RT-qPCR and Western Blot analyses in the erythroid progeny of edited SCD HSPCs confirmed α-globin downregulation at RNA and protein levels. Lastly, we combined both strategies using ABEs to target simultaneously BS within the BCL11A and MCS-R2 enhancers in SCD HSPCs. The concomitant usage of two sgRNAs did not affect editing efficiency at the different targeted sites, which was ~80% at both BCL11A and MCS-R2 enhancers. The erythroid progeny of edited SCD HSPCs harboring mutations in both BCL11A and MCS-R2 enhancers showed downregulation of α-globin and up-regulation of γ-globin at RNA and protein expression level. We expect that this combined strategy will efficiently rescue the pathological phenotype of RBCs derived from both SCD and β-thalassemic HSPCs. In conclusion, our study identified the core nucleotides of both the BCL11A and HBA1/2 MCS-R2 enhancers, which are crucial for regulating the expression of these genes and represent potential targets for scarless editing through BE to finely modulate globin expression. We also developed efficient BE strategies to simultaneously reactivate HbF and downregulate the α-globin expression in patient HSPCs. Validation of the above-described results in HSPCs in vivo will provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies.

Construction of a cytidine base editor based on Exopalaemon carinicauda cytidine deaminase and its application in crustacean genome editing

Economic crustaceans play important roles in aquaculture and create great benefits for the world agricultural economy. Studies have found that there are many single nucleotide polymorphism (SNP) sites in the crustacean genomes, which closely related to a variety of life activities like growth, development, reproduction, and so on. However, the in vivo functional study of SNPs still lacks appropriate technique in crustaceans. The cytosine base editor (CBE) based on cytidine deaminase (CDA) can convert the C•G base pair to T•A base pair, which provides a new idea for the study of function of crustacean SNPs. In this study, the CDA gene from Exopalaemon carinicauda (EcCDA) was cloned and fused to the N-terminus of Cas9 nickase (nCas9(D10A)) skeleton to construct a cytidine base editor (pEcCDA-nCas9(D10A)) suitable for crustacean point editing. Neocaridina denticulata sinensis MIH (NdMIH) gene was selected as the target gene. The in vitro transcribed EcCDA-nCas9(D10A) mRNA and NdMIH-gRNA were co-injected into the 1-cell stage embryos of N. denticulata sinensis for gene editing. Detection results showed that the constructed pEcCDA-nCas9(D10A) successfully realized the conversion of C•G to T•A in NdMIH with the editing efficiency of approximately 11.21%. Our works construct a well-established crustacean base editing platform.

Global spectrum of USH2A mutation in inherited retinal dystrophies: Prompt message for development of base editing therapy.

PurposeMutation in the USH2A gene is the most common cause of inherited retinal dystrophy (IRD), including non-syndromic retinitis pigmentosa (RP) and Usher syndrome II (USH2). Gene editing and therapy targeting USH2A, especially the hotspot region, would benefit a large proportion of IRD patients. In this study, we comprehensively analyzed the genetic spectrum of the USH2A gene, aiming to identify global hot spot mutations in USH2A-related IRDs and differences in hot spot regions across continents.Materials and methodsA retrospective USH2A-related IRD study was conducted, including our IRD cohort, and reported USH2A studies worldwide.ResultsA total of 3,972 mutated USH2A alleles of approximately 1,935 patients were collected from 33 cohort studies worldwide, containing 102 alleles of 51 patients in our IRD cohort. Mutations in exon 13 were the most common, reaching 18.4% globally and a higher frequency of 22% in America, 19.2% in Europe, and a lower 12% in East Asia. Pathogenic mutations that affected 10 of the 72 exons of USH2A, exon 2, exon 13, exon 41–43, exon 50, exon 54, exon 57, exon 61, and exon 63 in total were responsible for half of global USH2A mutant alleles. With base editors including adenine base editor (ABE), cytidine base editor (CBE), and glycosylase base editor (GBE), 76.3% of single nucleotide variations (SNVs) and 58% of all mutations in USH2A are correctable. Meantime, four novel pathogenic mutations were revealed in our IRD cohort, p. (Val1130Cysfs*72), p. (Ala2139fs*14), p. (Gly4139Arg), and p. (Val4166Cysfs*7).ConclusionIn this study, we revealed four novel mutations, expanding the spectrum of USH2A mutations, and importantly presented global hotspot exons and mutations of USH2A as well as the proportion of SNVs that can be restored by different base editors, providing a perspective for exploring high-efficiency and broader-reaching gene editing and gene therapies.

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.