An optimized base editor with efficient C-to-T base editing in zebrafish
- PDF / 2,884,425 Bytes
- 9 Pages / 595.276 x 790.866 pts Page_size
- 40 Downloads / 162 Views
RESEARCH ARTICLE
Open Access
An optimized base editor with efficient C-to-T base editing in zebrafish Yu Zhao1, Dantong Shang1, Ruhong Ying1, Hanhua Cheng1* and Rongjia Zhou1,2*
Abstract 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. Keywords: CRISPR/Cas9, Base editor, BE4max, Zebrafish
Background A large number of human genetic diseases are caused by point mutations or disease-associated SNP variants [1]. Large-scale genome-wide association studies have identified many loci that are associated with key human traits and diseases, for example, lifespan and aging [2]. In recent years, deep whole-genome sequencing of human populations generated numerous databases of genetic variations, including SNPs of pathogenic variants. Mutation spectrum analysis indicated that major mutations were C:G>T:A and T:A>C:G transitions associated with metabolic traits and disorders [3]. Although some of * Correspondence: [email protected]; [email protected] 1 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China Full list of author information is available at the end of the article
these mutations have been determined as causal or pathogenic variants, many disease-associated SNP variants remain to be defined by functional investigations. Zebrafish has become a popular model species for the study of gene function [4, 5]. Genome sequencing showed that approximately 70% of human genes have at least one obvious zebrafish ortholog [5]. Gene mutations in zebrafish can indeed mimic many human diseases, for example, ATP-sensitive potassium channel mutation for genetic cardiovascular disorder [6] and otx2b mutatio
Data Loading...
