Rationally Designed Base Editors for Precise Editing of the Sickle Cell Disease Mutation

Chu, Su; Packer, Michael S.; Rees, Holly A.; Lam, Dieter K.; Yu, Yi; Marshall, Jeffrey; Cheng, Lo-I; Lam, Daisy; Olins, Jenny; Ran, F. Ann; Liquori, Alexander; Gantzer, Bob; Decker, Jeremy; Born, David A.; Barrera, Luis; Hartigan, Adam J.; Gaudelli, Nicole M.; Ciaramella, Giuseppe; Slaymaker, Ian M.

doi:10.1089/crispr.2020.0144

Cited by 69 publications

(47 citation statements)

References 29 publications

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“…Importantly, engraftment capacity of base edited HSPCs in immunodeficient mice was not altered in comparison with uneditied cells either with from HD CB and mPB CD34+ cells were used. These results are consistent with recent studies performed in healthy and sickle cell HSPCs 41, 48, 49 . Further studies will directly address the important question of engraftment potential for base edited FA cells.…”

Section: Discussionsupporting

confidence: 94%

Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Siegner

Clemens

Ugalde

et al. 2022

Preprint

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Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions lead to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from an FA patient was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

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Section: Discussionsupporting

confidence: 94%

Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Siegner

Clemens

Ugalde

et al. 2022

Preprint

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“…Indeed, we observed obviously reduced off-target editing of both molecules in two of our inlaid Sa-CBEs. Consistent with this observation, three recent reports also demonstrated that inlaid SaCas9 and SpCas9 base editors showed decreased Cas9-independent RNA or DNA off-target editing 37 - 39 . In our R-loop assay, we noted that the editing efficiency for each cytosine in the artificial R-loop varied significantly, possibly because of the different accessibilities between the editable cytosines and the deaminase.…”

Section: Discussionsupporting

confidence: 72%

Internally inlaid SaCas9 base editors enable window specific base editing

Jiang¹,

Long²,

Yang³

et al. 2022

Theranostics

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Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.

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“…First, structural analyses of Nme2Cas9 [84] indicate that the position of the N-terminally fused TadA8e domain relative to the predicted path of the displaced strand is not optimal. With other effectors, domain-inlaid deaminase fusions have proven to be advantageous in some contexts [29,54], and the same is likely to be true with Nme2-ABEs. Second, the current Nme2-ABE8e has a wide editing window that could result in increased bystander editing.…”

Section: Discussionmentioning

confidence: 99%

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

Zhang

Bamidele

et al. 2021

Preprint

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Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

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Rationally Designed Base Editors for Precise Editing of the Sickle Cell Disease Mutation

Cited by 69 publications

References 29 publications

Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Internally inlaid SaCas9 base editors enable window specific base editing

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

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