scholarly journals Precise adenine base editors that exhibit minimized cytosine catalysis

2020 ◽  
Author(s):  
You Kyeong Jeong ◽  
SeokHoon Lee ◽  
Gue-Ho Hwang ◽  
Sung-Ah Hong ◽  
Se-eun Park ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Adenosine Deaminase ◽  
Rna Editing ◽  
The Other ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Target Dna ◽  
Editing Activity ◽  
Target Rna ◽  
Adenine Base

Abstract Adenine base editors (ABEs) promise specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target DNA site and exhibit transcriptome-wide off-target RNA editing. To alleviate the ABE-mediated cytosine editing activity, here we engineered the commonly-used version of adenosine deaminase, TadA7.10, to contain rationally designed mutations. We ultimately found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited greatly reduced cytosine deamination activity, and conversely, ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity rather than adenine editing. We found that the D108Q mutation also reduces cytosine deamination activity in two recently-developed versions of ABE, ABE8e and ABE8s, and has a synergistic effect with V106W, a key mutation that reduces off-target RNA editing. On the other hand, by incorporating the P48R mutation into ABE7.10, we demonstrated TC-specific base editing tools that enable either TC-to-TT or TC-to-TG conversions, broadening the utility of base editors.

scholarly journals Analysis and minimization of cellular RNA editing by DNA adenine base editors

2019 ◽  
Vol 5 (5) ◽  
pp. eaax5717 ◽  
Author(s):  
Holly A. Rees ◽  
Christopher Wilson ◽  
Jordan L. Doman ◽  
David R. Liu
Keyword(s):  
Rna Editing ◽  
Product Formation ◽  
Base Pairs ◽  
Base Editing ◽  
Dna And Rna ◽  
Mammalian Cell Lines ◽  
Target Dna ◽  
Editing Activity ◽  
Dna Editing ◽  
Adenine Base

Adenine base editors (ABEs) enable precise and efficient conversion of target A•T base pairs to G•C base pairs in genomic DNA with a minimum of by-products. While ABEs have been reported to exhibit minimal off-target DNA editing, off-target editing of cellular RNA by ABEs has not been examined in depth. Here, we demonstrate that a current ABE generates low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in both deaminase domains, we developed new ABE variants that retain their ability to edit DNA efficiently but show greatly reduced RNA editing activity, as well as lower off-target DNA editing activity and reduced indel by-product formation, in three mammalian cell lines. By decoupling DNA and RNA editing activities, these ABE variants increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity.

scholarly journals Efficient precise in vivo base editing in adult dystrophic mice

2020 ◽  
Author(s):  
Li Xu ◽  
Chen Zhang ◽  
Haiwen Li ◽  
Peipei Wang ◽  
Yandi Gao ◽  
...  
Keyword(s):  
Rna Editing ◽  
Deep Sequencing ◽  
High Efficiency ◽  
Contractile Function ◽  
Functional Improvement ◽  
Base Editing ◽  
Monogenic Diseases ◽  
Editing Activity ◽  
Target Rna

ABSTRACTBackgroundRecent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.MethodsWe employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.ResultsWe showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.ConclusionsThis study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.

scholarly journals Classification of CRISPR/Cas system and its application in tomato breeding

2022 ◽  
Author(s):  
Abira Chaudhuri ◽  
Koushik Halder ◽  
Asis Datta
Keyword(s):  
Protein Composition ◽  
Base Editing ◽  
Detailed Classification ◽  
Target Dna ◽  
Class 1 ◽  
Mechanisms Of Adaptation ◽  
Tomato Breeding ◽  
Primary Focus ◽  
Target Rna

AbstractRemarkable diversity in the domain of genome loci architecture, structure of effector complex, array of protein composition, mechanisms of adaptation along with difference in pre-crRNA processing and interference have led to a vast scope of detailed classification in bacterial and archaeal CRISPR/Cas systems, their intrinsic weapon of adaptive immunity. Two classes: Class 1 and Class 2, several types and subtypes have been identified so far. While the evolution of the effector complexes of Class 2 is assigned solely to mobile genetic elements, the origin of Class 1 effector molecules is still in a haze. Majority of the types target DNA except type VI, which have been found to target RNA exclusively. Cas9, the single effector protein, has been the primary focus of CRISPR-mediated genome editing revolution and is an integral part of Class 2 (type II) system. The present review focuses on the different CRISPR types in depth and the application of CRISPR/Cas9 for epigenome modification, targeted base editing and improving traits such as abiotic and biotic stress tolerance, yield and nutritional aspects of tomato breeding.

scholarly journals Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Minh Thuan Nguyen Tran ◽  
Mohd Khairul Nizam Mohd Khalid ◽  
Qi Wang ◽  
Jacqueline K. R. Walker ◽  
Grace E. Lidgerwood ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Dna ◽  
Dna Editing ◽  
Target Activity ◽  
Adenine Base

Abstract Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

scholarly journals BEON: A Functional Fluorescence Reporter for Quantification and Enrichment of Adenine Base-Editing Activity

2020 ◽  
Vol 28 (7) ◽  
pp. 1696-1705 ◽  
Author(s):  
Peipei Wang ◽  
Li Xu ◽  
Yandi Gao ◽  
Renzhi Han
Keyword(s):  
Base Editing ◽  
Editing Activity ◽  
Adenine Base

scholarly journals Next-generation cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity

2020 ◽  
Author(s):  
Yi Yu ◽  
Thomas Leete ◽  
David A. Born ◽  
Lauren Young ◽  
Luis A. Barrera ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Point Mutations ◽  
Next Generation ◽  
Dna Double Strand Breaks ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Dna And Rna ◽  
Fold Reduction ◽  
Target Dna ◽  
Target Activity

Abstract/introductory paragraphCytosine base editors (CBEs) are molecular machines which enable efficient and programmable reversion of T•A to C•G point mutations in the human genome without induction of DNA double strand breaks1, 2. Recently, the foundational cytosine base editor (CBE) ‘BE3’, containing rAPOBEC1, was reported to induce unguided, genomic DNA3, 4 and cellular RNA5 cytosine deamination when expressed in living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput cellular assay to select next-generation CBEs that display reduced spurious deamination profiles relative to rAPOBEC1-based CBEs, whilst maintaining equivalent or superior on-target editing frequencies. We screened 153 CBEs containing cytidine deaminase enzymes with diverse sequences and identified four novel CBEs with the most promising on/off target ratios. These spurious-deamination-minimized CBEs (BE4 with either RrA3F, AmAPOBEC1, SsAPOBEC3B, or PpAPOBEC1) were further optimized for superior on- and off-target DNA editing profiles through structure-guided mutagenesis of the deaminase domain. These next-generation CBEs display comparable overall DNA on-target editing frequencies, whilst eliciting a 10- to 49-fold reduction in C-to-U edits in the transcriptome of treated cells, and up to a 33-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Taken together, these next-generation CBEs represent a new collection of base editing tools for applications in which minimization of spurious deamination is desirable and high on-target activity is required.

scholarly journals Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

2020 ◽  
Author(s):  
Dhruva Katrekar ◽  
Nathan Palmer ◽  
Yichen Xiang ◽  
Anushka Saha ◽  
Dario Meluzzi ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Motifs ◽  
Mutagenesis Screen ◽  
Base Editing ◽  
Adenosine Deaminases ◽  
Editing Activity ◽  
Exogenous Delivery ◽  
Deaminase Domain ◽  
The Impact ◽  
Increased Activity

ABSTRACTAdenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5’ guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5’-GA-3’ motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

scholarly journals Adenovirus VAI RNA Antagonizes the RNA-Editing Activity of the ADAR Adenosine Deaminase

Virology ◽  
1998 ◽  
Vol 245 (2) ◽  
pp. 188-196 ◽  
Author(s):  
Ming Lei ◽  
Yong Liu ◽  
Charles E. Samuel
Keyword(s):  
Adenosine Deaminase ◽  
Rna Editing ◽  
Editing Activity

scholarly journals CRISPR adenine and cytosine base editors with reduced RNA off-target activities

2019 ◽  
Author(s):  
Julian Grünewald ◽  
Ronghao Zhou ◽  
Sowmya Iyer ◽  
Caleb A. Lareau ◽  
Sara P. Garcia ◽  
...  
Keyword(s):  
Rna Editing ◽  
Cytidine Deaminase ◽  
Single Nucleotide ◽  
Cytidine Deaminases ◽  
Guide Rna ◽  
Adenosine Deaminases ◽  
Dna Base ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Rna

AbstractCRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies5. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing5. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

scholarly journals Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jianan Li ◽  
Wenxia Yu ◽  
Shisheng Huang ◽  
Susu Wu ◽  
Liping Li ◽  
...  
Keyword(s):  
Rna Editing ◽  
Potential Interaction ◽  
Reporter System ◽  
Independent Manner ◽  
Robust Detection ◽  
Guide Rna ◽  
Highly Efficient ◽  
Adenine Base

AbstractBoth adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.

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