Adenine base editor engineering reduces editing of bystander cytosines

Jeong, You Kyeong; Lee, Seok-Hoon; Hwang, Gue‐Ho; Hong, Sung-Ah; Park, Se eun; Kim, Jin‐Soo; Woo, Jae Sung; Bae, Sangsu

doi:10.1038/s41587-021-00943-2

Cited by 69 publications

(49 citation statements)

References 27 publications

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“…Base editors evolved with various engineered versions of Tad 22 . Thus, we compared the A3-to-G3 conversion rates for all Tad variants developed thus far and found that the architectures of the codon-optimized Tad-Tad* (V106W, D108Q) 23,24 showed the highest conversion rates compared to any of the other forms (Supplementary Fig. 3a, Supplementary Table 1).…”

Section: Feasibility Of Dtnpb-based Adenine Base Editorsmentioning

confidence: 99%

Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA

Kim¹,

Chung²,

Lee

et al. 2022

Nat Chem Biol

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Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1 due to its high sequence similarity. Previously, we reported a set of engineered guide RNAs supporting high indel e ciency for Cas12f1 in human cells. Here, we suggest a new technology whereby the engineered gRNAs also manifest highly e ciency programmable endonuclease activity for TnpB, termed TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors. A codon-optimized Tad-Tad mutant (V106W, D108Q) dimer fused to the C-terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded by either developing several PAM variants of TnpB or by engineering TnpB and optimizing deaminases at PAM-distal and PAM-proximal sites, respectively. When delivered by AAV, the TaRGET-ABE showed potent A-to-G conversion rates in human cells. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by AAV, thereby harnessing the development of CRISPR-based gene therapy. MainLiving organisms are subjected to spontaneous genetic variations, which form the basis for biodiversity and evolution. The random nature of genetic variation as well as non-biological factors are also responsible for a variety of genetic disorders. Of the types of genetic variations identi ed in humans, single-nucleotide variations (SNVs) account for nearly half of disease-related mutations 1 . This suggests that the development of site-speci c, precise genome editing tools holds promise for the treatment of otherwise intractable genetic disorders. CRISPR/Cas-mediated base-editing systems have addressed the unsatisfactory modi cation e ciency of homology-directed repair (HDR) by exploiting the high genetargeting capability of the CRISPR/Cas systems. The catalytically inactive Cas (dCas) or nickase Cas (nCas) fused to naturally occurring or engineered deaminases led to highly e cient single-nucleotide alterations including the C:G-to-T:A 2 and A:T-to-G:C 3 conversion. The introduction of an E. coli-derived uracil DNA N-glycosylase also enabled C:G-to-G:C transversion 4 . Moreover, the base editing systems guarantee higher levels of safety from a clinical view point because they enable precise genome editing with negligible or low levels of double-strand breaks (DSBs). In addition to such innate high e ciency and safety, base editing systems have further evolved in various aspects including decreased off-target editing, enhanced conversion speci city, broadened editing windows, increased editing e ciency, and control of unwanted editing [5][6][7] .Despite the dramatic improvements of base-editing system per se, the challenges on the delivery side remain a major hurdle preventing base editors from being widely used in clinical applications 8 . Adenoassociated viruses (AAVs) are considered as a validated delivery platform due to the...

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Section: Feasibility Of Dtnpb-based Adenine Base Editorsmentioning

confidence: 99%

Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA

Kim¹,

Chung²,

Lee

et al. 2022

Nat Chem Biol

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“…3F). This result indicates that, to reduce the ABE-mediated C editing, further engineering of ABE would be necessary ( 35 ).…”

Section: Resultsmentioning

confidence: 99%

High-purity production and precise editing of DNA base editing ribonucleoproteins

Jang

Lee

et al. 2021

Sci. Adv.

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Ribonucleoprotein (RNP) complex–mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector–mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM–targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.

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“…1b). On the third target site previously used for evaluation of cytosine bystander mutations 13,16 , ABEmax and ABE8e induced lots of cytosine mutations (8.83% and 45.20% in average) while V28F, V28N, N108Q, L145C, L145T and L145Q mutations exhibited high A-to-G activity on A4 with greatly reduced cytosine conversions (ranging from 2.43% to 11.47%) (Fig. 1c).…”

Section: Structure-based Molecular Evolution Of Tada-8ementioning

confidence: 98%

“…The copyright holder for this preprint this version posted August 13, 2022. ; https://doi.org/10.1101/2022.08.12.503700 doi: bioRxiv preprint the increase of deamination activity, ABE8e exhibits significant Cas9-independent DNA and RNA off-target editing 9,13,14 .…”

Section: Introductionmentioning

confidence: 99%

“…ABE8e and ABE8s also showed quite high editing efficiencies in the livers of mice and non-human primates 11 or hemopoietic stem cells from sickle cell anemia patients 12 , demonstrating their glorious potential for gene therapeutics. However, with the increase of deamination activity, ABE8e exhibits significant Cas9-independent DNA and RNA off-target editing 9, 13, 14 .…”

Section: Introductionmentioning

confidence: 99%

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Engineering precise adenine base editor with infinitesimal rates of bystander mutations and off-target editing

Chen

Zhang

Xue

et al. 2022

Preprint

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Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise the concerns of safety issues. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2nt with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision than ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Due to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic SNVs when fused to Cas9 variants with expanded PAM compatibility.

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Adenine base editor engineering reduces editing of bystander cytosines

Cited by 69 publications

References 27 publications

Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA

Hypercompact adenine base editors based on a Cas12f variant guided by engineered RNA

High-purity production and precise editing of DNA base editing ribonucleoproteins

Engineering precise adenine base editor with infinitesimal rates of bystander mutations and off-target editing

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