Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

Chen, Yuxi; Zhi, Shengyao; Liu, Weiliang; Wen, Jinkun; Hu, Sihui; Cao, Tianqi; Sun, Hongwei; Li, Yang; Huang, Li; Liu, Yizhi; Liang, Puping; Huang, Junjiu

doi:10.1002/smtd.202000309

Cited by 42 publications

(26 citation statements)

References 63 publications

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“…Using the compact PE, we screened 4 split sites combined with Npu intein. Consistent with previous studies using base editors, we found split-cPE2-573 showed the highest on-target editing efficiency among split sites at endogenous loci 3,8,19 . Furthermore, we also found the compact PE without RNase H domain has no interaction with eRF1 which maintains the basal level of stop codon readthrough.…”

Section: Discussionsupporting

confidence: 92%

“…(split-cPE2-637), Q674 (split-cPE2-674), or Q713 (split-cPE2-713) 3,8,19,20 . To determine the effect of each split site on cPE2 fusion, we co-transfected the N-terminal and C-terminal fragments of split-PE2 variants into HEK293T cells and measured protein expression using western blot.…”

Section: Development Of Efficient Split-pementioning

confidence: 99%

“…The smaller size of cPE2 allows Cas9 to be split up to residue L551 from the C-terminal (Supplementary Fig. 4a), enabling the use of the potentially more efficient E573 Cas9 split site 3,19 .…”

Section: Development Of Efficient Split-pementioning

confidence: 99%

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Development of a flexible split prime editor using truncated reverse transcriptase

Xue

Zheng

Liang

et al. 2021

Preprint

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Prime Editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9, which affects editing efficiency, is limited due to the large size of PE. Furthermore, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.

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Section: Discussionsupporting

confidence: 92%

Section: Development Of Efficient Split-pementioning

confidence: 99%

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Development of a flexible split prime editor using truncated reverse transcriptase

Xue

Zheng

Liang

et al. 2021

Preprint

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“…To this aim, a suitable in vivo delivery system, such as AAV or HDAd5/35++ adenovirus vectors should be used. So far, several studies have established a system to deliver in vivo ABEs or CBEs using AAV vectors (Winter et al, 2019 ; Chen et al, 2020 ; Hu et al, 2020 ; Levy et al, 2020 ). As BEs are large enzymes that cannot be packaged in a single AAV, many groups used a split-intein system based on the use of two AAV vectors harboring the two moieties of a SpCas9-based CBE fused to intein fragments that are reassembled in vivo via trans -splicing.…”

Section: Challenges Of Base Editing Approachesmentioning

confidence: 99%

Base and Prime Editing Technologies for Blood Disorders

2021

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Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches–including the delivery of base editors and the off-target events–and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

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“…In conventional genome editing, smaller CRISPR ortholog Staphylococcus aureus (Sa) Cas9 has led to the development of single AAV vector 12,13 , but adding a deaminase domain has been di cult 14 . Instead of composing a single AAV vector, the base editing components could be split into two AAV vectors to circumvent the size limitation 15 .…”

Section: Introductionmentioning

confidence: 99%

Cytosine base editing systems with minimized off-target effect and molecular size

Li¹,

Mitsunobu²,

Yoshioka³

et al. 2021

Preprint

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Structure-based rational engineering of the cytosine base editing system Target-AID was performed to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 was eliminated and additional mutations were introduced to restore enzyme function. The resulting tCDA1EQ was effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized gRNA-independent off-targets, as assessed in yeast and human cells. Combining with the smaller Cas9 ortholog system, the smallest cytosine base editing system was created that is within the size limit of AAV vector.

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Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

Cited by 42 publications

References 63 publications

Development of a flexible split prime editor using truncated reverse transcriptase

Development of a flexible split prime editor using truncated reverse transcriptase

Base and Prime Editing Technologies for Blood Disorders

Cytosine base editing systems with minimized off-target effect and molecular size

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