Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants

Yang, Lei; Zhang, Xiaohui; Wang, Liren; Yin, Shuming; Zhu, Biyun; Xie, Ling; Duan, Qiuhui; Hu, Huiqiong; Zheng, Rui; Wu, Yu; Peng, Liangyue; Han, Hongyu; Zhang, Jiqin; Qiu, Wenjuan; Geng, Hui; Siwko, Stefan; Zhang, Xueli; Liu, Mingyao; Li, Dali

doi:10.1007/s13238-018-0568-x

Cited by 80 publications

(58 citation statements)

References 18 publications

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“…Although ABE variants recognizing non-NGG PAMs have been described [12][13][14] , editing efficiencies of these constructs are decreased in many instances when compared to outcomes observed with S. pyogenes Cas9 targeting NGG PAM sequences [12][13][14] . To determine whether our eighth-generation evolved deaminase also increases the editing efficiencies at target sites bearing non-NGG PAMs, we created ABE8 editors that replace S. pyogenes Cas9 with an engineered S. py.…”

Section: Abe8s With Either Non-ngg Pam Ncas9 Variants or Catalyticallmentioning

confidence: 99%

“…2c, Supplementary Figs. [14][15][16]. Encouragingly, dC9-ABE8 variants only have a median 14% reduction in on-target DNA editing efficiencies relative to nickase-active ABE8s.…”

Section: Abe8s With Either Non-ngg Pam Ncas9 Variants or Catalyticallmentioning

confidence: 99%

“…To date, seventh generation ABEs (ABE7) have enabled efficient A•T to G•C conversion in the genomes of humans 5 , mice [6][7][8] , bacteria 1 , plants 9,10 , and a variety of other species, reviewed here 11 . Many therapeutic targets, however, may benefit from a more active ABE with a broader editing window or improved compatibility with non-NGG nCas9s [12][13][14] as well as increased editing efficiencies in human cell lines 15 or when used in vivo 8 . The need for a more active version of ABE7.10 is the greatest when target adenines are positioned on the outer edges of the canonical ABE editing window (positions 3, 4, 7 and 8).…”

mentioning

confidence: 99%

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Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli

Lam

Rees

et al. 2020

Preprint

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The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ∼1.5x higher editing at protospacer positions A5-A7 and ∼3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ∼4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

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Section: Abe8s With Either Non-ngg Pam Ncas9 Variants or Catalyticallmentioning

confidence: 99%

“…2c, Supplementary Figs. [14][15][16]. Encouragingly, dC9-ABE8 variants only have a median 14% reduction in on-target DNA editing efficiencies relative to nickase-active ABE8s.…”

Section: Abe8s With Either Non-ngg Pam Ncas9 Variants or Catalyticallmentioning

confidence: 99%

mentioning

confidence: 99%

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Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli

Lam

Rees

et al. 2020

Preprint

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“…Based on evolutionary of CBE, ABE was also developed to expand the editing scope by using natural and engineered Cas9 variants with PAM specificities (Table ; Hu et al, ; Hua, Tao, & Zhu, ; L. Yang et al, ). Studies have shown that expression levels of base editors are major bottlenecks for base‐editing efficiency.…”

Section: Current Base‐editing Technology In Genomic Dnamentioning

confidence: 99%

“…Thus, the use of base editors is still limited, even though natural and engineered Cas9 variants with different PAM specificities have been developed (Hu et al, ; Hua et al, ; Y. B. Kim et al, ; Koblan et al, ; L. Yang et al, ). Moreover, the deamination window of base editors mainly depends on which deaminase is fused.…”

Section: Challenges Of Base Editorsmentioning

confidence: 99%

Single‐nucleotide editing: From principle, optimization to application

2019

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Cytosine base editors (CBEs) and adenine base editors (ABEs), which are generally composed of an engineered deaminase and a catalytically impaired CRISPR-Cas9 variant, are new favorite tools for single base substitution in cells and organisms. In this review, we summarize the principle of base-editing systems and elaborate on the evolution of different platforms of CBEs and ABEs, including their deaminase, Cas9 variants, and editing outcomes. Moreover, we highlight their applications in mouse and human cells and discuss the challenges and prospects of base editors. The ABEand CBE systems have been used in gene silencing, pathogenic gene correction, and functional genetic screening. Single base editing is becoming a new promising genetic tool in biomedical research and gene therapy.

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Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7

Price

Cruz

Bryson

et al. 2020

Biotech & Bioengineering

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The CRISPR‐Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram‐positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach‐through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR‐based editing mode of action in B. subtilis providing evidence that the nuclease‐mediated DNA double‐strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.

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Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants

Cited by 80 publications

References 18 publications

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Single‐nucleotide editing: From principle, optimization to application

Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7

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