Precise A•T to G•C base editing in the zebrafish genome

Qin, Wenzhi; Lu, Xiaochan; Liu, Yunxing; Bai, Haipeng; Li, Song; Lin, Shuo

doi:10.1186/s12915-018-0609-1

Cited by 42 publications

(31 citation statements)

References 26 publications

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“…Recently, base editing is a developing approach that can efficiently introduce point mutations in a programmable way without inducing double-strand breaks (DSBs) [9]. Until now, two classes of DNA base editor-cytosine base editor (CBE) and adenine base editor (ABE)-have been described and widely used in plants and animals, including zebrafish [10][11][12][13][14]. In addition, several strategies were developed to expand the base editors, such as a narrowed editing window, expanded targeting scope [15], improved editing specificity [16], and changed protospacer adjacent motif (PAM) compatibilities [15].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…Cytosine base editors (CBEs) could be used for converting the C•G base pair to a T•A base pair, and adenine base editors (ABEs) convert the A•T base pair to a G•C base pair within a window of several nucleotides at a target site [ 9 , 10 ]. However, undesirable byproducts such as small insertions or deletions (indels) in cells [ 13 , 14 ], animals [ 15 ], plants [ 16 ], and other organisms [ 17 , 18 , 19 ] have been verified in recent studies. Moreover, unexpected off-target effects have been widely reported in genome-editing cells using both CBE and ABE systems [ 20 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects

Liang

Sui

Liu

et al. 2020

Cells

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The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly “shut off” nuclease activity of both CBE and ABE systems.

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“…Previous studies have shown that optimization of genome editing nucleases for their specific use in zebrafish using codon optimization has been effective in improving their efficiencies [ 24 , 26 , 53 ]. Zebrafish codon-optimized versions of adenine base editors ABE7.10 (zABE7.10) and ABEmax (zABE7.10max), where both adenosine deaminase and nCas9 were replaced with their zebrafish codon-optimized counterparts (zTadA, zTadA* and znCas9), showed significantly increased editing at several loci as compared with the original ABE7.10 [ 26 ]. Thus, we can speculate that similar optimization of BE4max and AncBE4max base editors would increase their efficiencies in the zebrafish.…”

Section: Discussionmentioning

confidence: 99%

“…Recently developed programmable base editing technology that combines the targeting specificity of Cas9 with a cytidine or adenine deaminase provides an alternate to HDR for generation of zebrafish with C:G to T:A or A:T to G:C substitutions [ 3 , 23 , 24 , 25 , 26 , 27 , 28 ]. In a typical base editor, nCas9 is fused to cytidine or adenine deaminase with small linkers [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

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

Carrington

Weinstein

2020

Cells

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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.

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Precise A•T to G•C base editing in the zebrafish genome

Cited by 42 publications

References 26 publications

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

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

AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects

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

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