scholarly journals EditR: A novel base editing quantification software using Sanger sequencing

2017 ◽  
Author(s):  
Mitchell G. Kluesner ◽  
Derek A. Nedveck ◽  
Walker S. Lahr ◽  
John R. Garbe ◽  
Juan E. Abrahante ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Cytidine Deaminase ◽  
Cost Effective ◽  
Enzymatic Assays ◽  
Online Tool ◽  
Base Editing ◽  
Guide Rna ◽  
Desktop Application ◽  
Fusion Enzymes ◽  
Position Type

ABSTRACTCRISPR/Cas9-Cytidine deaminase fusion enzymes - termed Base Editors – allow targeted editing of genomic deoxcytidine to deoxthymidine (C→T) without the need for double stranded break induction. Base editors represent a paradigm-shift in gene editing technology, due to their unprecedented efficiency to mediate targeted, single-base conversion; however, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time consuming. To overcome these limitations, we developed a simple, cost effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed EditR. We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence (baseeditr.com). EditR is more accurate than enzymatic assays, and provides added insight to the position, type and efficiency of base editing. Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

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.

scholarly journals MultiEditR: An easy validation method for detecting and quantifying RNA editing from Sanger sequencing

2019 ◽  
Author(s):  
Mitchell Kluesner ◽  
Annette Arnold ◽  
Taga Lerner ◽  
Rafail Nikolaos Tasakis ◽  
Sandra Wüst ◽  
...  
Keyword(s):  
Rna Editing ◽  
Sanger Sequencing ◽  
Tumour Progression ◽  
Cost Effective ◽  
Base Change ◽  
Rna Seq ◽  
Multiple Sources ◽  
Base Editing ◽  
Cost Effective Method ◽  
Apobec Family

ABSTRACTRNA editing is the base change that results from RNA deamination by two predominant classes of deaminases; the APOBEC family and the ADAR family. Respectively, deamination of nucleobases by these enzymes are responsible for endogenous editing of cytosine to uracil (C-to-U) and adenosine to inosine (A-to-I). RNA editing is known to play an essential role both in maintaining normal cellular function, as well as altered cellular physiology during oncogenesis and tumour progression. Analysis of RNA editing in these important processes, largely relies on RNA-seq technology for the detection and quantification of RNA editing sites. Despite the power of these technologies, multiple sources of error in detecting and measuring base editing still exist, therefore additional validation and quantification of editing through Sanger sequencing is still required for confirmation of editing. Depending on the number of RNA editing sites that are of interest, this validation step can be both expensive and time-consuming. To address this need we developed the tool MultiEditR which provides a simple, and cost-effective method of detecting and quantifying RNA editing form Sanger sequencing. We expect that MultiEditR will foster further discoveries in this rapidly expanding field.

scholarly journals Base editing using CRISPR/Cas9 in Drosophila

2021 ◽  
Author(s):  
Elizabeth Marr ◽  
Christopher J. Potter
Keyword(s):  
Cytidine Deaminase ◽  
Point Mutations ◽  
Tissue Expression ◽  
Genomic Region ◽  
Target Sequence ◽  
Molecular Scaffolds ◽  
Base Editing ◽  
Guide Rna ◽  
Somatic Tissues ◽  
Transgenic Lines

AbstractCas9 and a guide RNA function to target specific genomic loci for generation of a double stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase to induce C>T (or G>A) mutations at targeted sites. Here, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (DBE2, based on the mammalian BE2) which consists of a fusion protein of cytidine deaminase, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila Base Editor (Actin5C-DBE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C>T were found in positions 3-9 of the gRNA targeted site, with conversion reaching nearly 100% of targeted DNA is somatic tissues. The simultaneous use of two gRNA targeting a genomic region spaced ∼50 bps apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.

scholarly journals Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti

2021 ◽  
Vol 12 ◽  
Author(s):  
Longxiang Wang ◽  
Yuan Xiao ◽  
Xiaowei Wei ◽  
Jimin Pan ◽  
Deqiang Duanmu
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Cytidine Deaminase ◽  
Base Editing ◽  
Guide Rna ◽  
Genetic Manipulations ◽  
Highly Efficient ◽  
Adenine Deaminase ◽  
Nucleotide Resolution ◽  
Biological Nitrogen

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.

scholarly journals CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

2020 ◽  
Author(s):  
Efthymia Symeonidi ◽  
Julian Regalado ◽  
Rebecca Schwab ◽  
Detlef Weigel
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Repair Process ◽  
Wild Type ◽  
Isochorismate Synthase ◽  
Guide Rna ◽  
Cost Effective Method ◽  
Variable Efficiency

AbstractBackgroundGenome editing with the CRISPR/Cas9 system allows the user to mutate a targeted region of the genome using an endonuclease (Cas9) and an artificial single-guide RNA (sgRNA). Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to CRISPR/Cas9 mutagenesis. This process can be laborious, expensive and inefficient with conventional methods such as the T7E1 assay or Sanger sequencing. An alternative comprises methods for amplicon sequencing, but most available protocols do not include a facile way for high throughput generation of the samples for sequencing.ResultsIn this study we provide a full pipeline based on amplicon sequencing, CRISPR-finder. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. For example, we were able to analyze in one sequencing reaction over 900 Arabidopsis thaliana individuals whose genomes had been targeted with the CRISPR/Cas9 system.ConclusionsIn order to validate the potential of CRISPR-finder, we targeted the ISOCHORISMATE SYNTHASE 1 gene in A. thaliana using the CRISPR/Cas9 system. We successfully identified a mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and -efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

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

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.

scholarly journals The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Minkyung Choi ◽  
Jae-Young Yun ◽  
Jun-Hyuk Kim ◽  
Jin-Soo Kim ◽  
Sang-Tae Kim
Keyword(s):  
Arabidopsis Thaliana ◽  
Cytidine Deaminase ◽  
Biological Research ◽  
Loss Of Function ◽  
Nucleotide Substitutions ◽  
Viral Origin ◽  
Nonsense Mutations ◽  
Base Editing ◽  
Low Efficiency ◽  
Viable System

AbstractCRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

scholarly journals ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Jingke Xie ◽  
Xingyun Huang ◽  
Xia Wang ◽  
Shixue Gou ◽  
Yanhui Liang ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Multiple Point ◽  
Nucleotide Polymorphisms ◽  
Spacer Length ◽  
Endogenous Gene ◽  
Base Editing ◽  
Fetal Fibroblasts

Abstract Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

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