Glycosylase base editors enable C-to-A and C-to-G base changes

De, Zhao; Li, Ju; Li, Siwei; Xin, Xiuqing; Hu, Muzi; Price, Marcus A.; Rosser, Susan J.; Bi, Chen

doi:10.1038/s41587-020-0592-2

Cited by 293 publications

(176 citation statements)

References 32 publications

(38 reference statements)

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“…Some existing strategies could be used to expand the targeting scope of ACBE because previous studies have developed many variants of CRISPR-Cas protein requiring different PAM sequences or the restricted-less PAMs [31,32]. Recently, C-to-G base editors consisted of a Cas9 nickase, a cytidine deaminase variant, and uracil DNA N-glycosylase (eUNG), which could induce targeted C-to-G transversions with a high editing specificity in mammalian cells [33,34]. Similarly, new dual-function base editors could be generated to simultaneously induce C-to-G and A-to-G by using the strategies established in this study.…”

Section: Discussionmentioning

confidence: 99%

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

et al. 2020

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Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

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Section: Discussionmentioning

confidence: 99%

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

et al. 2020

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“…Recently, novel forms of DNA base editing such as a C>G editing [15][16][17] , and simultaneous C and A editing [18][19][20][21] have been suggested, which would substantially expand the utilities of DNA base editors. Along with the intense efforts to improve DNA base editors, our suggested tools, high-delity ABE variants that exhibit minimized cytosine catalysis and reduced off-target RNA editing, and TC-speci c base editors with negligible bystander effects, should make DNA base editing tools a more attractive alternative for gene editing in many research areas, such as disease therapy development, gene regulation, and plant transformation.…”

mentioning

confidence: 99%

Precise adenine base editors that exhibit minimized cytosine catalysis

Jeong

Lee

Hwang

et al. 2020

Preprint

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Adenine base editors (ABEs) promise specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target DNA site and exhibit transcriptome-wide off-target RNA editing. To alleviate the ABE-mediated cytosine editing activity, here we engineered the commonly-used version of adenosine deaminase, TadA7.10, to contain rationally designed mutations. We ultimately found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited greatly reduced cytosine deamination activity, and conversely, ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity rather than adenine editing. We found that the D108Q mutation also reduces cytosine deamination activity in two recently-developed versions of ABE, ABE8e and ABE8s, and has a synergistic effect with V106W, a key mutation that reduces off-target RNA editing. On the other hand, by incorporating the P48R mutation into ABE7.10, we demonstrated TC-specific base editing tools that enable either TC-to-TT or TC-to-TG conversions, broadening the utility of base editors.

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“…By mutating the codons encoding amino acids to stop codons, the researchers successfully inactivated the corresponding genes in E. coli [18], C. glutamicum [85], Klebsiella pneumoniae [87], and Staphylococcus aureus [88], which proved that the CBE tool can achieve gene inactivation. At present, the ABE base editor is only used to realize A to G base changes in E. coli [84,89]. The biggest advantage of the base editor compared with CRISPR/Cas9-guided gene editing is that it does not cause bacterial death, but a major disadvantage is that it can only induce base replacement, leading to mutation or gene inactivation, and cannot be used for gene insertions or deletions.…”

Section: Crispr/dcas9-mediated Base Editormentioning

confidence: 99%

Application of different types of CRISPR/Cas-based systems in bacteria

et al. 2020

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As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12amediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.

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Glycosylase base editors enable C-to-A and C-to-G base changes

Cited by 293 publications

References 32 publications

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

Precise adenine base editors that exhibit minimized cytosine catalysis

Application of different types of CRISPR/Cas-based systems in bacteria

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