scholarly journals Author Correction: Engineered pegRNAs improve prime editing efficiency

2021 ◽  
Author(s):  
James W. Nelson ◽  
Peyton B. Randolph ◽  
Simon P. Shen ◽  
Kelcee A. Everette ◽  
Peter J. Chen ◽  
...  
Keyword(s):  
Editing Efficiency

scholarly journals Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 878
Author(s):  
Oskar Gustafsson ◽  
Julia Rädler ◽  
Samantha Roudi ◽  
Tõnis Lehto ◽  
Mattias Hällbrink ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Cell Penetrating Peptide ◽  
Wild Type ◽  
Efficient Manner ◽  
Editing Efficiency ◽  
Freeze Thaw ◽  
Enzyme Digest ◽  
Clinical Potential ◽  
Generation Sequencing

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

scholarly journals Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporationmediated delivery and embryo culture

MethodsX ◽  
2021 ◽  
pp. 101419
Author(s):  
Anil K Challa ◽  
Denise Stanford ◽  
Antonio Allen ◽  
Lawrence Rasmussen ◽  
Ferdinand K Amanor ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Gene Editing ◽  
Editing Efficiency

scholarly journals AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liyang Zhang ◽  
John A. Zuris ◽  
Ramya Viswanathan ◽  
Jasmine N. Edelstein ◽  
Rolf Turk ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Genome Editing ◽  
Nk Cell ◽  
Basic Research ◽  
Cell Recognition ◽  
Site Specific ◽  
Editing Efficiency ◽  
Rapid Generation ◽  
Therapeutic Cell

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

scholarly journals Automated and Manual Data Editing: A View on Process Design and Methodology

2013 ◽  
Vol 29 (4) ◽  
pp. 511-537 ◽  
Author(s):  
Jeroen Pannekoek ◽  
Sander Scholtus ◽  
Mark Van der Loo
Keyword(s):  
Process Design ◽  
A Priori ◽  
Empirical Evaluation ◽  
Efficiency Gains ◽  
Specific Nature ◽  
Editing Efficiency ◽  
Data Editing ◽  
Optimal Mix ◽  
Editing Process ◽  
Statistical Functions

Abstract Data editing is arguably one of the most resource-intensive processes at NSIs. Forced by everincreasing budget pressure, NSIs keep searching for more efficient forms of data editing. Efficiency gains can be obtained by selective editing, that is, limiting the manual editing to influential errors, and by automating the editing process as much as possible. In our view, an optimal mix of these two strategies should be aimed for. In this article we present a decomposition of the overall editing process into a number of different tasks and give an upto- date overview of all the possibilities of automatic editing in terms of these tasks. During the design of an editing process, this decomposition may be helpful in deciding which tasks can be done automatically and for which tasks (additional) manual editing is required. Such decisions can be made a priori, based on the specific nature of the task, or by empirical evaluation, which is illustrated by examples. The decomposition in tasks, or statistical functions, also naturally leads to reuseable components, resulting in efficiency gains in process design.

scholarly journals One-step multiple site-specific base editing by direct embryo injection for precision and pyramid pig breeding

2020 ◽  
Author(s):  
Ruigao Song ◽  
Yu Wang ◽  
Qiantao Zheng ◽  
Jing Yao ◽  
Chunwei Cao ◽  
...  
Keyword(s):  
Stop Codon ◽  
Point Mutations ◽  
Embryonic Cells ◽  
Site Specific ◽  
Editing Efficiency ◽  
Base Editing ◽  
One Step ◽  
Pig Performance ◽  
Novel Alleles

AbstractPrecise and simultaneous acquisition of multiple beneficial alleles in the genome to improve pig performance are pivotal for making elite breeders. Cytidine base editors (CBEs) have emerged as powerful tools for site-specific single nucleotide replacement. Here, we compare the editing efficiency of four CBEs in porcine embryonic cells and embryos to show that hA3A-BE3-Y130F and hA3A-eBE3-Y130F consistently results in higher base-editing efficiency and lower toxic effects to in vitro embryo development. We also show that zygote microinjection of hA3A-BE3-Y130F results in one-step generation of pigs (3BE pigs) harboring C-to-T point mutations, including a stop codon in CD163 and in MSTN and induce beneficial allele in IGF2. The 3BE pigs showed improved growth performance, hip circumference, food conversion rate. Our results demonstrate that CBEs can mediate high throughput genome editing by direct embryo microinjection. Our approach allows immediate introduction of novel alleles for beneficial traits in transgene-free animals for pyramid breeding.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

Genome editing's potential target diseases in the cardiovascular field

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Cardiovascular Diseases ◽  
Genome Editing ◽  
Target Cells ◽  
Clinical Use ◽  
Editing Efficiency ◽  
Potential Target ◽  
Genome Wide ◽  
Significant Barrier

Two types of cardiovascular diseases can be cured or prevented using genome editing. The liver is the organ that has received the most attention in terms of clinical genome editing for cardiovascular diseases. Off-target mutagenesis is a concern of any form of genome editing. Off-target mutations in target cells or tissues may lead to undesirable functional phenotypes, including cancer. For therapeutic editing of the heart, the authors claim it's critical to achieve high editing efficiency at a chosen genomic site in a desired tissue. Off-target editing can be tested genome-wide unbiasedly using newer cell-based methods. For low off-target impact, well-designed gRNAs are important. The delivery of genome editors to target tissues and cells is a significant barrier to clinical use.

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