Advances in base editing: A focus on base transversions

High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells

Adenine base editors (ABEs) and cytosine base editors (CBEs) enable the single nucleotide editing of targeted DNA sites avoiding generation of double strand breaks, however, the genomic features that influence the outcomes of base editing in vivo still remain to be characterized. High-throughput datasets from lentiviral integrated libraries were used to investigate the sequence features affecting base editing outcomes, but the effects of endogenous factors beyond the DNA sequences are still largely unknown. Here the base editing outcomes of ABE and CBE were evaluated in mammalian cells for 5012 endogenous genomic sites and 11,868 genome-integrated target sequences, with 4654 genomic sites sharing the same target sequences. The comparative analyses revealed that the editing outcomes of ABE and CBE at endogenous sites were substantially different from those obtained using genome-integrated sequences. We found that the base editing efficiency at endogenous target sites of both ABE and CBE was influenced by endogenous factors, including epigenetic modifications and transcriptional activity. A deep-learning algorithm referred as BE_Endo, was developed based on the endogenous factors and sequence information from our genomic datasets, and it yielded unprecedented accuracy in predicting the base editing outcomes. These findings along with the developed computational algorithms may facilitate future application of BEs for scientific research and clinical gene therapy.

RNA-DNA hybrid binding domain broadens the editing window of base editors.

Point mutations represent the majority of known human genetic variants associated with disease but are difficult to correct cleanly and efficiently using standard nuclease‐based genome editing methods. In this lecture I will describe the development, application, and evolution of base editing, a new approach to genome editing that directly converts a target base pair to another base pair in living cells without requiring DNA backbone cleavage or donor DNA templates. Through a combination of protein engineering and protein evolution, we recently developed two classes of base editors (BE4 and ABE) that together enable all four types of transition mutations (C to T, T to C, A to G, and G to A) to be efficiently and cleanly installed or corrected at target positions in genomic DNA. The four transition mutations collectively account for most known human pathogenic point mutations. Base editing has been widely used by many laboratories around the world in a wide range of organisms including bacteria, fungi, plants, fish, frogs, insects, mammals, and even human embryos. We have recently expanded the scope of base editing by enhancing its efficiency, product purity, targeting scope, and DNA specificity. By optimizing base editor expression, we developed “max” versions of cytosine and adenine base editors with greatly increase editing efficiency in mammalian cells. We also show that base editing can function in vivo in post‐mitotic somatic cells that do not support homology‐directed repair. To improve the targeting scope of base editing, we used our phage‐assisted continuous evolution (PACE) system to rapidly evolve Cas9 and base editor variants with both broadened PAM compatibility and higher DNA specificity. Finally, we integrated several of these developments to address cell and animal models of human genetic disease. Base editing can be used to correct pathogenic point mutations, introduce disease‐suppressing mutations, and create new models of genetic diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

Development of a Simple and Quick Method to Assess Base Editing in Human Cells

CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC‐based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, and editing CC and GC motifs is challenging and inefficient. To address these challenges, existing tRNA adenine deaminase (TadA)‐derived CBEs are evaluated in zebrafish, and a series of zTadCBE variants is developed that demonstrate high editing efficiency, minimized off‐target effects, and an expanded targeting range compared to existing tools. The approach integrates beneficial mutations from TadA‐based adenine base editors (ABEs) with SpRYCas9n‐enhanced protospacer‐adjacent motif (PAM) compatibility. The expanded window zTadCBE variants enable the targeting of cytosines at a broader range of nucleotide positions relative to the PAM sequence, further enhancing the versatility of this tool. Using zTadCBEs, four zebrafish disease models affecting the auditory, nervous, metabolic, and muscular systems are generated directly in the F0 generation—models that cannot be efficiently produced using earlier CBE tools. Together, zTadCBE variants provide a robust and flexible toolkit for efficient and precise C‐to‐T base editing in zebrafish, facilitating rapid in vivo functional assessment of genetic variants.

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.

The power and versatility of genome editing tools in crop improvement

Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing

SummaryBase editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology‐directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.

Base editing (BE) is a powerful tool for engineering single-nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids, and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, unlike Cas9, which shows remarkable efficacy in creating homozygous disruptive mutations following DNA double-strand breaks (DSBs), BE is relatively inefficient and identifying and isolating edited cells for analysis has proved challenging. To enable simple identification and enrichment of base editing events, we developed a “Gene On” (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We validate GO using an activatable GFP and use it to measure the kinetics, efficiency, PAM specificity, and fidelity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes, or enzymes such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. We further demonstrate the application of GO in detecting in vivo BE activity using a newly generated mouse model with inducible and reversible base editor expression that will enable generation of novel cancer models in vivo. GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research. This abstract is also being presented as Poster A45. Citation Format: Alyna Katti, Miguel Foronda, Jill Zimmerman, Bianca Diaz, Maria Paz Zafra, Sukanya Goswami, Elena Piskounova, Lukas E. Dow. Strategies to improve engineering cancer-associated SNVs with base editing [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHA05.

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1–4. However, we recently demonstrated that they can also induce widespread guide RNA-independent RNA base edits5 and created SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that have reduced unwanted RNA editing activity5. Here, we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA editing and comparable on-target DNA activities that are also among the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A (hA3A)-based CBE induces substantial RNA base edits, whereas an enhanced A3A (eA3A)-CBE6, human activation-induced cytidine deaminase (hAID)-CBE7, and the petromyzon marinus cytidine deaminase (pmCDA1)-based CBE Target-AID4 induce reduced RNA edits. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base editor coding sequences.

Papaya is a major tropical fruit crop with notable nutritional and economic value, yet its genetic improvement through modern breeding technologies faces substantial challenges. The traditional tissue culture process is both labor-intensive and time-consuming, causing gene editing advancements in papaya to lag behind those in other crops. To overcome these obstacles, we developed a tissue culture–independent hairy root system in papaya, which enables efficient gene editing and significantly enhances the application and development of editing tools. This innovative platform allows for the pre-assessment of editing efficiency and supports the establishment of adenine base editor (ABE) and cytosine base editor (CBE) tools in papaya, thereby mitigating the high failure costs associated with the lengthy cycle of conventional genetic transformation. Utilizing this system, we pre-tested sgRNA activity and achieved high editing efficiency of CpWIP3 during stable transformation. Additionally, through promoter screening, we successfully developed ABE and CBE tools, marking the first precise single-nucleotide editing system in papaya. This gene-editing system provides a crucial platform for advancing functional genomics and accelerating precision breeding in papaya.

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.