High-efficiency plastome base-editing in rice with TAL cytosine deaminase

AbstractEfficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.

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Genome Editing in Agriculture: Technical and Practical Considerations

International Journal of Molecular Sciences ◽

10.3390/ijms20122888 ◽

2019 ◽

Vol 20 (12) ◽

pp. 2888 ◽

Cited By ~ 8

Author(s):

Julia Jansing ◽

Andreas Schiermeyer ◽

Stefan Schillberg ◽

Rainer Fischer ◽

Luisa Bortesi

Keyword(s):

Genome Editing ◽

Mechanism Of Action ◽

High Efficiency ◽

Public Acceptance ◽

Crop Species ◽

Base Editing ◽

The Public ◽

Intact Plants ◽

Efficient Delivery ◽

Selection Of

The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to different outcomes, but also have implications for the public acceptance and regulatory approval of genome-edited plants. Despite the high efficiency and precision of the tools, there are still major bottlenecks in the generation of new and improved varieties, including the efficient delivery of the genome-editing reagents, the selection of desired events, and the regeneration of intact plants. In this review, we evaluate current delivery and regeneration methods, discuss their suitability for important crop species, and consider the practical aspects of applying the different genome-editing techniques in agriculture.

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Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

10.1101/2020.05.20.103614 ◽

2020 ◽

Author(s):

Xi Xiang ◽

Kunli Qu ◽

Xue Liang ◽

Xiaoguang Pan ◽

Jun Wang ◽

...

Keyword(s):

High Throughput ◽

Gene Editing ◽

High Efficiency ◽

Outcome Data ◽

Massively Parallel ◽

Human Embryonic Kidney Cells ◽

Editing Efficiency ◽

Base Editing ◽

Target Sites ◽

In Cells

AbstractThe CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

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Simultaneous high-efficiency base editing and reprogramming of patient fibroblasts

Stem Cell Reports ◽

10.1016/j.stemcr.2021.10.017 ◽

2021 ◽

Author(s):

Sami Jalil ◽

Timo Keskinen ◽

Rocío Maldonado ◽

Joonas Sokka ◽

Ras Trokovic ◽

...

Keyword(s):

High Efficiency ◽

Base Editing

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Efficient precise in vivo base editing in adult dystrophic mice

10.1101/2020.06.24.169292 ◽

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.

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Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

10.1101/2020.12.12.422520 ◽

2020 ◽

Author(s):

Marion Rosello ◽

Juliette Vougny ◽

François Czarny ◽

Marina Mione ◽

Jean-Paul Concordet ◽

...

Keyword(s):

High Efficiency ◽

Human Gene ◽

Point Mutations ◽

Base Change ◽

Model System ◽

Human Genetic Disease ◽

Base Editing ◽

Oncogenic Mutations ◽

Cell Signaling Pathways

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes among which cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

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216 Multiplex base editing of NK cell to enhance cancer immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.216 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A229-A229

Author(s):

Minjing Wang ◽

Mitchell Kluesner ◽

Patricia Claudio Vázquez ◽

Beau Webber ◽

Branden Moriarity

Keyword(s):

T Cells ◽

Nk Cells ◽

Cancer Immunotherapy ◽

High Efficiency ◽

Cell Function ◽

Nk Cell ◽

Functional Assay ◽

Single Base ◽

Editing Efficiency ◽

Base Editing

BackgroundNatural killer (NK) cells have many unique features that have gained attention in cancer immunotherapy. NK cells can kill in antigen independent and dependent fashion, can be used as an allogeneic product, and perform antibody-dependent cell-mediated cytotoxicity (ADCC). However, NK cell function is regulated by many activating and inhibitory receptors, which cancer cells take advantage of to avoid being killed by NK cells. NK cells are also known for their technical and biological challenges which result in low editing efficiencies, compared to T cells and other immune cells.MethodsBase editing (BE) is a CRISPR-Cas9 based genome editing technology that allows precise single base transitions. Previously, we reported a high efficiency method for multiplex engineering of T cells using BE and thus reasoned that applying similar concepts in NK cells may offer an opportunity to alter many genes simultaneously at higher efficiency through multiplex base editing. We thus selected a panel of genes bearing critical roles in NK cell function for immunotherapy, including inhibitory intracellular regulator AHR and CISH, inhibitory checkpoint receptor KLRG1, TIGIT, KLRC1, and PDCD1, and Fc receptor CD16A. CD16A is responsible for NK cell ADCC and is regulated via cleavage upon NK activation. Non-cleavable CD16A improves ADCC killing and can be achieved through single-base substitution with BE.ResultsUsing the adenosine BE (ABE8e), we achieved multiplex editing (6 genes) rates up to 99% and 95% editing/knockout at DNA and protein levels, respectively. Notably, we assessed for reduction in editing efficiency when additional genes were targeted and found no significant reduction in editing efficiencies when targeting up to 6 genes simultaneously. Moreover, functional evaluation of non-cleavable CD16A NK cells revealed up to 35% increase of cytotoxicity against Raji cells.ConclusionsWe were able to achieve high multiplex editing efficiency in primary human NK cells using ABE8eand there was no significant decrease of editing efficiency as the number of gene of interest increases, up to 6 genes in total. Functional assay confirmed increased NK cell cytotoxicity against tumor cells. Our end goal is to achieve high efficiency multiplex editing in CAR-expressing NK cells to further improve NK cell activity and toxicity for cancer immunotherapy.ReferenceWebber B, Lonetree C, Kluesner M, et al. Highly efficient multiplex human T cell engineering without double-strand breaks usingCas9 base editors. Nat Commun 2019;10:5222.

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Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

eLife ◽

10.7554/elife.65552 ◽

2021 ◽

Vol 10 ◽

Author(s):

Marion Rosello ◽

Juliette Vougny ◽

François Czarny ◽

Marina C Mione ◽

Jean-Paul Concordet ◽

...

Keyword(s):

High Efficiency ◽

Human Gene ◽

Point Mutations ◽

Base Change ◽

Model System ◽

Human Genetic Disease ◽

Base Editing ◽

Oncogenic Mutations ◽

Cell Signaling Pathways

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

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Recent Advances in CRISPR/Cas9 Directed Base Editing

Current Gene Therapy ◽

10.2174/1566523221666210322090638 ◽

2021 ◽

Vol 21 ◽

Author(s):

Nan Liu ◽

Lifang Zhou ◽

Junyan , Qu ◽

Shaohua Yao

Keyword(s):

Recent Progress ◽

High Efficiency ◽

Genetic Diseases ◽

Point Mutations ◽

Clinical Applications ◽

Great Promise ◽

Base Editing ◽

Recent Advances ◽

Dna Base ◽

Genomic Editing

: Recently, CRISPR based techniques had significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled irreversible conversion of specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, high efficiency of those editing tools also provided great promise in clinical applications. In this review, we discuss recent progress and challenges of base editing tools.

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