Sequence-specific prediction of the efficiencies of adenine and cytosine base editors

Song, Ming; Kim, Hui Kwon; Lee, Sungtae; Kim, Younggwang; Seo, Sangyeon; Park, Jinman; Choi, Jae Woo; Jang, Hyewon; Shin, Jeong Hong; Min, Shungeng; Quan, Zhejiu; Kim, Ji Hun; Kang, Hoon Chul; Yoon, Sungroh

doi:10.1038/s41587-020-0573-5

Cited by 90 publications

(96 citation statements)

References 50 publications

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“…Additionally, Cas9-CPs without DNA cleavage activities but retaining DNA binding abilities remained to be discovered and evaluated in the future, as our results suggested that under specific conditions, DNA cleavage activity might be dispensable for SpCas9-DSs-based tools. Additionally, modest positive correlations have been observed between N-terminal-based conventional ABE/CBE activities and DNA cleavage activity of Cas9 24 while ABE-nSpCas9-DS770 to ABE-nSpCas9-DS919 described here maintained high editing activities without any DNA cleavage activity. As regions 770-919 mainly cover the HNH domain in Cas9, it is possible that deaminase inside here might induce specific conformation changes to promote base editing process.…”

Section: Discussionmentioning

confidence: 64%

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

Yuan

Cao

et al. 2020

Nat Commun

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Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.

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Section: Discussionmentioning

confidence: 64%

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

Yuan

Cao

et al. 2020

Nat Commun

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“…Thus, for both base editors, the sequences in their editing windows and the bases surrounding the edited bases were kept unaltered. Neighboring (+/-1) nucleotides can strongly in uence the e ciency of base editing; 'GAC' and 'ACA' employed here for ABE and CBE, respectively, have been shown to be associated with medium level activities for both base editors 46 . Lacking data suggesting the opposite, we have expected that the differences in the 34 target sequences (in the PAM proximal 10 nucleotides) should primarily affect the interactions between the fused SpCas9 nuclease partner of the base editors and the targets, thus this experimental design was speci cally suited to study how the binding and cleavage propensities of SpCas9 variants affect the base editors' activities.…”

Section: On-target Activity Of Base Editors With Increased Delity Spcmentioning

confidence: 95%

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

Tálas

Simon

Kulcsár

et al. 2020

Preprint

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Adenine and cytosine base editors (ABE, CBE) are designed to generate single base mutations in genes without necessarily generating DNA double-strand breaks and undesired indel mutations. However, the activity of base editors employing an inactive (dead) SpCas9 is generally low, which may be increased only at the expense of generating undesired indels by using a nickase SpCas9. We have increased the efficiency of dead base editors to a level comparable to that of nickase base editors by enriching cells labelled for efficient base editing using Base Editor Activity Reporter (BEAR), a plasmid-based, fluorescent tool. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we have examined the applicability of increased fidelity variants to decrease Cas9-dependent off-target effects that revealed that CBE remains active on off-targets where increased fidelity mutations and/or mismatches decrease the activity of ABE, making the strategy of applying increased fidelity variants more beneficial for ABE than for CBE.

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“…To prepare pLenti-NG-PE2-BSD, the Lenti_Split-BE4-N-Blast plasmid 24 was digested with restriction enzymes AgeI and BamHI (New England Biolabs (hereafter, for brevity, NEB)) and a MEGAquick-spin total fragment DNA purification kit (iNtRON Biotechnology) was used to gel purify the linearized plasmid. Fragments of PE2-encoding sequence (Addgene #132775) and NG-Cas9encoding sequence (Addgene #124163) were amplified by PCR using Phusion Polymerase (NEB).…”

Section: Construction Of Plasmid Vectorsmentioning

confidence: 99%

Prime editing enables precise genome editing in mouse liver and retina

Jang

Shin

et al. 2021

Preprint

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Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

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Sequence-specific prediction of the efficiencies of adenine and cytosine base editors

Cited by 90 publications

References 50 publications

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

Prime editing enables precise genome editing in mouse liver and retina

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