Large-scale manufacturing of base-edited chimeric antigen receptor T cells.

Base editing is a revolutionary gene-editing technique enabling the introduction of point mutations into the genome without generating detrimental DNA double-stranded breaks. Base-editing enzymes are commonly delivered in the form of modified linear messenger RNA (mRNA) that is costly to produce. Here, we address this problem by developing a simple protocol for manufacturing base-edited cells using circular RNA (circRNA), which is less expensive to synthesize. Compared with linear mRNA, higher editing efficiencies were achieved with circRNA, enabling an 8-fold reduction in the amount of RNA required. We used this protocol to manufacture a clinical dose (1 × 108 cells) of base-edited chimeric antigen receptor (CAR) T cells lacking expression of the inhibitory receptor, PD-1. Editing efficiencies of up to 86% were obtained using 0.25 µg circRNA/1 × 106 cells. Increased editing efficiencies with circRNA were attributed to more efficient translation. These results suggest that circRNA, which is less expensive to produce than linear mRNA, is a viable option for reducing the cost of manufacturing base-edited cells at scale.

Optimization of the base editor BE4max in chicken somatic cells.

Advanced animal reproductive and breeding biotechnology has made it possible to alter traits or create new genetic resources by the direct knock-in or knock-out of target genes. Base editing technology can achieve single-base mutations without double-stranded DNA breaks, and is a promising tool for use in the genetic modification and breeding of livestock. However, the application of base editors (BEs) in chicken has not been optimized. We evaluated the efficacy of BE4max in chicken somatic cells (DF-1). The key element of BE4max, cytosine deaminase (APOBEC), was optimized for chicken. The base editing efficiency of the optimized chBE4max editor, compared with the original BE4max editor, was improved by 10.4% ± 4.6. By inhibiting the expression of the uracil DNA glycosylase-related gene methyl binding domain protein 4 (MBD4) by siRNA in chicken DF-1 cells, the editing efficiency was enhanced by 4.43% ± 1.4 compared to the control. These results suggest that this editor may have applications in poultry breeding studies.

Reduced off-target effect of NG-BE4max by using NG-HiFi system.

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.

An optimized base editor with efficient C-to-T base editing in zebrafish.

BACKGROUND: Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. RESULTS: Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). CONCLUSIONS: Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish.

The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.