BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

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.

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PEAR: a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

10.1101/2021.04.26.441486 ◽

2021 ◽

Author(s):

Dorottya A. Simon ◽

András Tálas ◽

Péter I. Kulcsár ◽

Ervin Welker

Keyword(s):

Single Cells ◽

Full Length ◽

Target Sequence ◽

Gene Engineering ◽

Fluorescent Reporter ◽

Background Fluorescence ◽

Sequence Variations ◽

Fundamental Research ◽

Editing Activity

ABSTRACTPrime editing is a recently developed gene engineering tool that allows the introduction of short insertions, deletions or substitutions into the genome. However, the efficiency of prime editing, generally reaching around 10-30% editing, has not resembled its versatility. Here, Prime Editor Activity Reporter (PEAR), a sensitive fluorescent tool is introduced for the identification of single cells with prime editing activity. Possessing no background fluorescence, PEAR specifically reports on prime editing events in individual cells. By design, it ensures unrestricted flexibility for sequence variations in the full length of the target sequence. The application of PEAR as an enrichment marker of prime editing can increase the edited population by up to 70% and alleviate the burden of the otherwise time and labour consuming process of cloning of the correctly edited cells, therefore considerably improving the applicability of prime editing in fundamental research and biotechnological uses.

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

Molecular Therapy ◽

10.1016/j.ymthe.2020.04.009 ◽

2020 ◽

Vol 28 (7) ◽

pp. 1696-1705 ◽

Cited By ~ 2

Author(s):

Peipei Wang ◽

Li Xu ◽

Yandi Gao ◽

Renzhi Han

Keyword(s):

Base Editing ◽

Editing Activity ◽

Adenine Base

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Precise adenine base editors that exhibit minimized cytosine catalysis

10.21203/rs.3.rs-84912/v1 ◽

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.

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GO: A functional reporter system to identify and enrich base editing activity

10.1101/862458 ◽

2019 ◽

Author(s):

Alyna Katti ◽

Miguel Foronda ◽

Jill Zimmerman ◽

Bianca Diaz ◽

Maria Paz Zafra ◽

...

Keyword(s):

Cell Lines ◽

Antibiotic Resistance Genes ◽

High Sensitivity ◽

Mouse Cell ◽

Single Nucleotide Variants ◽

Reporter System ◽

Base Editing ◽

Powerful Approach ◽

Editing Activity

ABSTRACTBase editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids, and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a “Gene On” (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency, and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes, or enzymes such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

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

10.1101/2020.09.08.288233 ◽

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.

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GO: a functional reporter system to identify and enrich base editing activity

Nucleic Acids Research ◽

10.1093/nar/gkaa124 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2841-2852 ◽

Cited By ~ 2

Author(s):

Alyna Katti ◽

Miguel Foronda ◽

Jill Zimmerman ◽

Bianca Diaz ◽

Maria Paz Zafra ◽

...

Keyword(s):

Cell Lines ◽

Antibiotic Resistance Genes ◽

High Sensitivity ◽

Mouse Cell ◽

Single Nucleotide Variants ◽

Reporter System ◽

Base Editing ◽

Powerful Approach ◽

Editing Activity

Abstract Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a ‘Gene On’ (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

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Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens

The FASEB Journal ◽

10.1096/fj.202001065rrr ◽

2020 ◽

Vol 34 (12) ◽

pp. 15907-15921

Author(s):

Kyung Youn Lee ◽

Hong Jo Lee ◽

Hee Jung Choi ◽

Soo Taek Han ◽

Kyu Hyuk Lee ◽

...

Keyword(s):

Base Excision Repair ◽

Germ Cells ◽

Excision Repair ◽

Primordial Germ Cells ◽

Repair Pathway ◽

Base Excision Repair Pathway ◽

Base Editing ◽

Editing Activity ◽

Base Excision

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A transient reporter for editing enrichment (TREE) in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz713 ◽

2019 ◽

Vol 47 (19) ◽

pp. e120-e120 ◽

Cited By ~ 8

Author(s):

Kylie Standage-Beier ◽

Stefan J Tekel ◽

Nicholas Brookhouser ◽

Grace Schwarz ◽

Toan Nguyen ◽

...

Keyword(s):

Fluorescent Protein ◽

Disease Modeling ◽

Cell Populations ◽

Base Editing ◽

Cell Enrichment ◽

A Cell ◽

Editing Activity ◽

Delivery Strategies ◽

Conventional Cell ◽

Green Fluorescent

Abstract Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.

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Predicting base editing outcomes using position-specific sequence determinants

10.1101/2021.09.16.460622 ◽

2021 ◽

Author(s):

Ananth Pallaseni ◽

Elin Madli Peets ◽

Jonas Koeppel ◽

Juliane Weller ◽

Luca Crepaldi ◽

...

Keyword(s):

Dna Sequences ◽

Level Control ◽

Specific Sequence ◽

Base Editing ◽

Guide Rna ◽

Machine Learning Model ◽

Efficient Planning ◽

Position Specificity ◽

Editing Activity ◽

The Impact

Nucleotide-level control over DNA sequences is poised to power functional genomics studies and lead to new therapeutics. CRISPR/Cas base editors promise to achieve this ability, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ~14,000 target sequences. Base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base, and is correlated with measures of Cas9 guide RNA efficiency. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, with a preceding thymine in both editor types leading to a wider editing window, while a preceding guanine in cytosine editors and preceding adenine in adenine editors to a narrower one. The impact of features on editing rate depends on the position, with guide RNA efficacy mainly influencing bases around the centre of the window, and sequence biases away from it. We use these observations to train a machine learning model to predict editing activity per position for both adenine and cytosine editors, with accuracy ranging from 0.49 to 0.72 between editors, and with better generalization performance across datasets than existing tools. We demonstrate the usefulness of our model by predicting the efficacy of potential disease mutation correcting guides, and find that most of them suffer from more unwanted editing than corrected outcomes. This work unravels the position-specificity of base editing biases, and provides a solution to account for them, thus allowing more efficient planning of base edits in experimental and therapeutic contexts.

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