Abstract Cytosine and adenosine base editors show great potential in research and clinical applications. Current iterations of the deaminase—the enzyme used to create precise single-nucleotide changes via base editing—exhibit various off-target effects, including Cas-independent off-targeting, off-base editing, and bystander editing. Engineered deaminases are typically derived from eukaryotic deaminases, which are larger and exhibit high levels of Cas-independent DNA editing, or from evolved variants of the E. coli TadA protein (ecTadA), which are smaller but frequently cause off-base editing. To overcome the limitations inherent to using a single protein sequence as the basis for engineering, we diversified 95 newly identified TadA orthologs by introducing literature-derived mutations and DNA shuffling to yield millions of training sequences for measuring base editor efficiency. Rather than pursuing multiple rounds of random mutagenesis and selection, we trained generative models on the performance data from the diversified pools of variants and drew on information-theoretic insights to efficiently explore the deaminase sequence space to generate diverse and high-performing deaminases. From a single round of diversification, we created a small set of novel and specific cytosine and adenosine deaminases that were markedly distinct in sequence from published base editor deaminases. We additionally found that the deaminases created by our model generally outperform those which we identified through typical directed evolution. The novel adenosine and cytosine deaminases identified in this work showed high on-base activity, comparable to the leading published base editors, but with demonstrably lower off-base activity. The cytosine deaminases were particularly compact compared to known sequences due to a truncation in their final α-helix.
12Development of a Simple and Quick Method to Assess Base Editing in Human Cells
Development of a Simple and Quick Method to Assess Base Editing in Human Cells
25Split complementation of base editors to minimize off-target edits.
Base editors (BEs) empower the efficient installation of beneficial or corrective point mutations in crop and human genomes. However, conventional BEs can induce unpredictable guide RNA (gRNA)-independent off-target edits in the genome and transcriptome due to spurious activities of BE-enclosing deaminases, and current improvements mostly rely on deaminase-specific mutagenesis or exogenous regulators. Here we developed a split deaminase for safe editing (SAFE) system applicable to BEs containing distinct cytidine or adenosine deaminases, with no need of external regulators. In SAFE, a BE was properly split at a deaminase domain embedded inside a Cas9 nickase, simultaneously fragmenting and deactivating both the deaminase and the Cas9 nickase. The gRNA-conditioned BE reassembly conferred robust on-target editing in plant, human and yeast cells, while minimizing both gRNA-independent and gRNA-dependent off-target DNA/RNA edits. SAFE also substantially increased product purity by eliminating indels. Altogether, SAFE provides a generalizable solution for BEs to suppress off-target editing and improve on-target performance.
Read more122The ADA Human Gene Therapy Clinical Protocol Original Covering Memo: February 23, 1990
Severe combined immunodeficiency (SCID) due to deficiency of the purine metabolic enzyme adenosine deaminase (ADA) is a fatal childhood immunodeficiency disease. Immune reconstitution by transplantation with HLA-identical bone marrow is the treatment of choice. For patients not candidates for bone marrow transplantation, we propose to attempt immune reconstitution by using infusions of autologous T lymphocytes expanded in tissue culture and genetically corrected by insertion of a normal ADA gene using retroviral-mediated gene transfer. The vector is LASN, in which the human ADA gene is promoted by the LTR while the NeoR gene is driven by the SV40 early gene promoter. The packaging line is PA317. The protocol is designed to have two parts. In Part 1, autologous gene-corrected T lymphocytes would be infused repeatedly in low numbers in order to build an immune repertoire of T cells and also to obtain information as to how long gene corrected T cells survive in vivo. In Part 2A, the gene-corrected T cells would be selected in G418 and/or 2'deoxyadenosine and reinfused into the patient at monthly intervals for approximately 6 months. The goals would be essentially the same as in Part 1. In Part 2B, the number of gene-corrected T cells would be escalated in half-log increments to the predicted therapeutic level (probably around 1 x 10(9)/kg). 1-3 x 10(9)/kg gene-corrected cells would be infused several times and the patients would be monitored in order to determine if significant clinical improvement has occurred.
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15Internally inlaid SaCas9 base editors enable window specific base editing
Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary.Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N.Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.
Read more73Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy.
Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.
Read more21Deep learning models incorporating endogenous factors beyond DNA sequences improve the prediction accuracy of base editing outcomes
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.
Read more10Precise base editing without unintended indels in human cells and mouse primary myoblasts
Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.
Read more55Site-directed RNA editing: recent advances and open challenges
RNA editing by cytosine and adenosine deaminases changes the identity of the edited bases. While cytosines are converted to uracils, adenines are converted to inosines. If coding regions of mRNAs are affected, the coding potential of the RNA can be changed, depending on the codon affected. The recoding potential of nucleotide deaminases has recently gained attention for their ability to correct genetic mutations by either reverting the mutation itself or by manipulating processing steps such as RNA splicing. In contrast to CRISPR-based DNA-editing approaches, RNA editing events are transient in nature, therefore reducing the risk of long-lasting inadvertent side-effects. Moreover, some RNA-based therapeutics are already FDA approved and their use in targeting multiple cells or organs to restore genetic function has already been shown. In this review, we provide an overview on the current status and technical differences of site-directed RNA-editing approaches. We also discuss advantages and challenges of individual approaches.
Read more12Design and Engineering of Light-Induced Base Editors Facilitating Genome Editing with Enhanced Fidelity.
Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double-stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off-target effects. Continuously expressed deaminases used for gene editing may lead to unwanted base alterations at unpredictable genomic locations. In the present study, blue-light-activated base editors (BLBEs) are engineered based on the distinct photoswitches magnets that can switch from a monomer to dimerization state in response to blue light. By fusing the N- and C-termini of split DNA deaminases with photoswitches Magnets, efficient A-to-G and C-to-T base editing is achieved in response to blue light in prokaryotic and eukaryotic cells. Furthermore, the results showed that BLBEs can realize precise blue light-induced gene editing across broad genomic loci with low off-target activity at the DNA- and RNA-level. Collectively, these findings suggest that the optogenetic utilization of base editing and optical base editors may provide powerful tools to promote the development of optogenetic genome engineering.
Read moreAnalysis of Cytosine Base Editors in Bovine Zygotes: Efficiency and Editing Window Characterization Through Targeting the MYO7A Gene
Cytosine base editors (CBEs) enable precise C-to-T (G-to-A) conversions in genomic DNA, offering significant potential for specific gene editing. This study compared the prototypical Base Editor 3 (BE3) and a modified variant, BE3-Y130F, which utilizes an hA3A deaminase with the Y130F mutation, focusing on their editing efficiency and editing window characteristics using bovine zygotes. Following in vitro fertilization (IVF), sgRNA and Cas9 mRNA were injected as a targeting efficiency control, which resulted in 100% editing with no wild-type sequence. Then, either BE3 or BE3-Y130F mRNA, synthesized via in vitro transcription, and an sgRNA targeting exon 4 of the MYO7A gene was injected into zygotes. Genomic DNA was extracted from both blastocysts and developmentally arrested embryos, and Sanger sequencing was performed to evaluate C-to-T conversion efficiency and editing window. Both BE3 and BE3-Y130F achieved 100% C-to-T conversion efficiency at the primary target cytosine. BE3 displayed a defined editing window, primarily affecting cytosines at positions 7 and 8, indicating a predictable profile. In contrast, BE3-Y130F maintained high efficiency but had a less clearly defined editing window, resulting in incomplete editing and a remaining cytosine on the target sequence.
Read more10The power and versatility of genome editing tools in crop improvement
The power and versatility of genome editing tools in crop improvement
1RNA-DNA hybrid binding domain broadens the editing window of base editors.
RNA-DNA hybrid binding domain broadens the editing window of base editors.
11Prediction of Base Editing Efficiencies and Outcomes Using DeepABE and DeepCBE.
Adenine base editors (ABEs) and cytosine base editors (CBEs) have been widely used to introduce disease-relevant point mutations at target DNA sites of interest. However, the introduction of point mutations using base editors can be difficult due to low editing efficiencies and/or the existence of multiple target nucleotides within the base editing window at the target site. Thus, previous works have relied heavily on experimentally evaluating the base editing efficiencies and outcomes using time-consuming and labor-intensive multi-step experimental processes. DeepABE and DeepCBE are deep learning-based computational models to predict the efficiencies and outcome frequencies of ABE and CBE at given target DNA sites, in silico. Here, we describe the step-by-step procedure for the accurate determination of specific target nucleotides for ABE or CBE editing on the online available web tool, (DeepBaseEditor, https://deepcrispr.info/DeepBaseEditor ).
Read more2Rationally Designed TadA‐Derived Cytosine Editors Enable Context‐Independent Zebrafish Genome Editing
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.
Read more17High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells
High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells
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