Generation of a multi-herbicide-tolerant alfalfa by using base editing

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

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CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

Electronic Journal of Biotechnology ◽

10.1016/j.ejbt.2021.04.010 ◽

2021 ◽

Author(s):

Salah Adlat ◽

Farooq Hayel ◽

Ping Yang ◽

Yang Chen ◽

Zin Mar Oo ◽

...

Keyword(s):

Cytosine Deaminase ◽

Base Editing

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Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽

10.3390/v13071288 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1288

Author(s):

Wendy Dong ◽

Boris Kantor

Keyword(s):

Large Scale ◽

Lentiviral Vector ◽

Clinical Applications ◽

Scale Production ◽

Base Editing ◽

Epigenome Editing ◽

Vector Systems ◽

Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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Advances in base editing with an emphasis on an AAV-based strategy

Methods ◽

10.1016/j.ymeth.2021.03.015 ◽

2021 ◽

Author(s):

Jiajie Kuang ◽

Qinghua Lyu ◽

Jiao Wang ◽

Yubo Cui ◽

Jun Zhao

Keyword(s):

Base Editing

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New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

International Journal of Molecular Sciences ◽

10.3390/ijms22010319 ◽

2020 ◽

Vol 22 (1) ◽

pp. 319

Author(s):

Jaiana Malabarba ◽

Elisabeth Chevreau ◽

Nicolas Dousset ◽

Florian Veillet ◽

Julie Moizan ◽

...

Keyword(s):

Target Genes ◽

Cytidine Deaminase ◽

Acetolactate Synthase ◽

Nucleotide Substitutions ◽

Discrete Variation ◽

Adventitious Regeneration ◽

Base Editing ◽

Perennial Plants ◽

Guide Rnas ◽

Albino Phenotype

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

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APOE: The New Frontier in the Development of a Therapeutic Target towards Precision Medicine in Late-Onset Alzheimer’s

International Journal of Molecular Sciences ◽

10.3390/ijms22031244 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1244

Author(s):

Anna Yang ◽

Boris Kantor ◽

Ornit Chiba-Falek

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Precision Medicine ◽

Antisense Oligonucleotides ◽

State Of The Art ◽

Late Onset ◽

Base Editing ◽

Medical Need ◽

New Frontier ◽

Amyloid Cascade

Alzheimer’s disease (AD) has a critical unmet medical need. The consensus around the amyloid cascade hypothesis has been guiding pre-clinical and clinical research to focus mainly on targeting beta-amyloid for treating AD. Nevertheless, the vast majority of the clinical trials have repeatedly failed, prompting the urgent need to refocus on other targets and shifting the paradigm of AD drug development towards precision medicine. One such emerging target is apolipoprotein E (APOE), identified nearly 30 years ago as one of the strongest and most reproduceable genetic risk factor for late-onset Alzheimer’s disease (LOAD). An exploration of APOE as a new therapeutic culprit has produced some very encouraging results, proving that the protein holds promise in the context of LOAD therapies. Here, we review the strategies to target APOE based on state-of-the-art technologies such as antisense oligonucleotides, monoclonal antibodies, and gene/base editing. We discuss the potential of these initiatives in advancing the development of novel precision medicine therapies to LOAD.

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Mapping CRISPR-Cas9 public and commercial innovation using The Lens institutional toolkit

Transgenic Research ◽

10.1007/s11248-021-00237-y ◽

2021 ◽

Author(s):

Osmat Azzam Jefferson ◽

Simon Lang ◽

Kenny Williams ◽

Deniz Koellhofer ◽

Aaron Ballagh ◽

...

Keyword(s):

Public Investment ◽

Open Data ◽

Regulatory Elements ◽

Policy Makers ◽

Base Editing ◽

The Public ◽

Homology Directed Repair ◽

A Genome ◽

Institutional Capability ◽

Cellular Dna

AbstractCRISPR-Cas9 is a revolutionary technology because it is precise, fast and easy to implement, cheap and components are readily accessible. This versatility means that the technology can deliver a timely end product and can be used by many stakeholders. In plant cells, the technology can be applied to knockout genes by using CRISPR–Cas nucleases that can alter coding gene regions or regulatory elements, alter precisely a genome by base editing to delete or regulate gene expression, edit precisely a genome by homology-directed repair mechanism (cellular DNA), or regulate transcriptional machinery by using dead Cas proteins to recruit regulators to the promoter region of a gene. All these applications can be for: 1) Research use (Non commercial), 2) Uses related product components for the technology itself (reagents, equipment, toolkits, vectors etc), and 3) Uses related to the development and sale of derived end products based on this technology. In this contribution, we present a prototype report that can engage the community in open, inclusive and collaborative innovation mapping. Using the open data at the Lens.org platform and other relevant sources, we tracked, analyzed, organized, and assembled contextual and bridged patent and scholarly knowledge about CRISPR-Cas9 and with the assistance of a new Lens institutional capability, The Lens Report Builder, currently in beta release, mapped the public and commercial innovation pathways of the technology. When scaled, this capability will also enable coordinated editing and curation by credentialed experts to inform policy makers, businesses and private or public investment.

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