Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain

Zhang, Xiaohui; Liang, Chen; Zhu, Biyun; Wang, Liren; Chen, Caiyu; Hong, Mengjia; Huang, Yifan; Li, Huiying; Han, Hongyu; Cai, Bailian; Yu, Weishi; Yin, Shuming; Yang, Lei; Yang, Zuozhen; Liu, Meizhen; Zhang, Ying; Mao, Zhiyong; Wu, Yuxuan; Liu, Mingyao; Li, Dali

doi:10.1038/s41556-020-0518-8

Cited by 79 publications

(90 citation statements)

References 49 publications

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“…To test whether the addition of a DBD in PE2 would enhance the prime-editing efficiency, we first added either Rad51 DBD or RPA70-C, both of which are ssDBDs that have previously been shown to increase the efficiency of cytosinebase editors 13 , between the Cas9 nickase and RT domains of PE2, generating PE2 variants named PE2-mid_Rad51 (hyperPE2 or for brevity, hyPE2) and PE2-mid_RPA70, respectively (Fig. 1a).…”

Section: Development Of Hype2mentioning

confidence: 99%

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

et al. 2021

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Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency < 1%), hyPE2 enables prime editing with efficiencies ranging from 1.1% to 2.9% at up to 34% of target sequences, potentially facilitating prime editing applications.

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Section: Development Of Hype2mentioning

confidence: 99%

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

et al. 2021

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“…Worth mentioning is a different kind of innovative application of ABEs or CBEs, the production of precise short deletions, as demonstrated in rice and wheat (Li et al 2020c;Wang et al 2020b). Another intriguing achievement is the dramatically enhanced efficiency of BE by fusion of single stranded DNA binding domain from Rad51 (Zhang et al 2020), which increases the accessibility of the substrate to the deaminase. However, it should be tested whether this type of fusion will lead to an increase of non-specific mutations in plants.…”

Section: Advances Of the Base Editing Techniquementioning

confidence: 99%

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Huang

Puchta

2021

Transgenic Res

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In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

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“…These variants were used to generate CP-BE4max enzymes that can efficiently edit bases that otherwise would be inaccessible (Huang et al, 2019 ). Interestingly, the introduction of the RAD51 single-stranded DNA binding domain (ssDBD) in BE4max, dramatically increased the editing frequency and extended the editing window to cytosines in positions 9–15 (hyBE4max) (Zhang et al, 2020a ). Similar results were obtained by inserting the RAD51 ssDBD in BE4max harboring APOBEC3A (hyA3A-BE4max) (Zhang et al, 2020a ).…”

Section: Development Of Cytosine and Adenine Base Editorsmentioning

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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Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain

Cited by 79 publications

References 49 publications

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Base and Prime Editing Technologies for Blood Disorders

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