scholarly journals PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1029 ◽  
Author(s):  
Rodrigo Ribeiro Arnt Sant’Ana ◽  
Clarissa Alves Caprestano ◽  
Rubens Onofre Nodari ◽  
Sarah Zanon Agapito-Tenfen
Keyword(s):  
Exposure Time ◽  
Dna Sequences ◽  
Mutation Frequency ◽  
Gene Editing ◽  
Inositol Phosphate ◽  
Gc Content ◽  
Assay Method ◽  
Ribonucleoprotein Complexes ◽  
Target Sites ◽  
Selection Of

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.

scholarly journals PEG-delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-editing Screening of Maize Protoplasts

2020 ◽  
Author(s):  
Rodrigo Ribeiro Arnt Sant’Ana ◽  
Clarissa Alves Caprestano ◽  
Rubens Onofre Nodari ◽  
Sarah Zanon Agapito-Tenfen
Keyword(s):  
Exposure Time ◽  
Dna Sequences ◽  
Mutation Frequency ◽  
Gene Editing ◽  
Inositol Phosphate ◽  
Gc Content ◽  
Assay Method ◽  
Ribonucleoprotein Complexes ◽  
Target Sites ◽  
Selection Of

CRISPR-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach and it can generate unintended transgenic integrations, while Cas9 prolonged expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. These downfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro- transcribed guide RNA (gRNA) molecules in a protoplast system. We therefore aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with PEG 4000. We performed effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene and applying two different exposure times to RNPs. Using low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between exposure time and mutation frequency. Mutation frequency was gRNA sequence- and exposure time-dependent. In summary, we demonstrated the suitability of RNP transfection as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for selection of gRNA suitable for editing of gene of interest will be highly useful for genome editing in maize, since genome size and GC-content are large and high in maize genome, respectively. Nevertheless, the large amplitude of mutations at target site requires scrutiny when checking mutations at off-target sites and potential safety concerns.

scholarly journals Is selecting better than modifying? An investigation of arguments against germline gene editing as compared to preimplantation genetic diagnosis

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Alix Lenia v. Hammerstein ◽  
Matthias Eggel ◽  
Nikola Biller-Andorno
Keyword(s):  
Dna Sequences ◽  
Reproductive Medicine ◽  
Gene Editing ◽  
Genetic Diseases ◽  
Genetic Diagnosis ◽  
Third Party ◽  
Human Embryos ◽  
Germline Gene ◽  
Future Child ◽  
Selection Of

Abstract Background Recent scientific advances in the field of gene editing have led to a renewed discussion on the moral acceptability of human germline modifications. Gene editing methods can be used on human embryos and gametes in order to change DNA sequences that are associated with diseases. Modifying the human germline, however, is currently illegal in many countries but has been suggested as a ‘last resort’ option in some reports. In contrast, preimplantation genetic (PGD) diagnosis is now a well-established practice within reproductive medicine. Both methods can be used to prevent children from being born with severe genetic diseases. Main text This paper focuses on four moral concerns raised in the debate about germline gene editing (GGE) and applies them to the practice of PGD for comparison: Violation of human dignity, disrespect of the autonomy and the physical integrity of the future child, discrimination of people living with a disability and the fear of slippery slope towards immoral usage of the technology, e.g. designing children for specific third party interests. Our analysis did not reveal any fundamental differences with regard to the four concerns. Conclusion We argue that with regard to the four arguments analyzed in this paper germline gene editing should be considered morally (at least) as acceptable as the selection of genomes on the basis of PGD. However, we also argue that any application of GGE in reproductive medicine should be put on hold until thorough and comprehensive laws have been implemented to prevent the abuse of GGE for non-medical enhancement.

Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases

2019 ◽  
Vol 88 (1) ◽  
pp. 191-220 ◽  
Author(s):  
Daesik Kim ◽  
Kevin Luk ◽  
Scot A. Wolfe ◽  
Jin-Soo Kim
Keyword(s):  
Dna Sequences ◽  
Gene Editing ◽  
Genomic Rearrangements ◽  
Zinc Finger Nucleases ◽  
Collateral Damage ◽  
Transcription Activator ◽  
Genome Wide ◽  
Target Sites ◽  
Programmable Nucleases ◽  
Target Activity

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

scholarly journals Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Constitutive Expression ◽  
Gc Content ◽  
Seed Region ◽  
Research Topics ◽  
Target Region ◽  
Cas9 Nuclease ◽  
Different Types ◽  
Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

scholarly journals A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

2004 ◽  
Vol 9 (4) ◽  
pp. 343-353 ◽  
Author(s):  
Elfrida R. Benjamin ◽  
Sarah L. Haftl ◽  
Dimitris N. Xanthos ◽  
Gregg Crumley ◽  
Mohamed Hachicha ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Column Chromatography ◽  
Large Volume ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Assay Method ◽  
Signal To Noise ◽  
Chromatography Method ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

scholarly journals First Complete Genome Sequence of Brucella abortus 2308 isolated from an abortion storm in a dairy farm in India

2021 ◽  
Author(s):  
Amit Kumar ◽  
Malyaj R Prajapati ◽  
Surendra Upadhyay ◽  
Anamika Bhordia ◽  
Vinod Kumar Singh ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Dna Sequences ◽  
Brucella Abortus ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Messenger Rna ◽  
Gc Content ◽  
Dairy Farm ◽  
Rrna Genes ◽  
Sequence Length

Abstract The present report communicates the first complete genome sequence of Brucella abortus 2308 strain isolated from a an abortion storm in a dairy farm located at Kanpur, Uttar Pradesh in India. It caused the last trimester abortions of 32 animals out of 100 cows in a dairy over a period of 60 days. The bacteria were isolated in pure culture from the placenta of aborted cows. The genome sequence length of isolated bacteria is 3,285,606 bp with a 57.25 % GC content, an N50 value of 296,426, L50 value of 4 containing 3,119 coding DNA sequences (CDSs), 49 tRNAs, 1 transfer messenger RNA (mRNA), and 3 rRNA genes. It is the first report of Brucella abortus 2308 isolation and complete genome sequence from Indian subcontinent.

scholarly journals Identification of Gene Related to Hard Bunch Phenotype in Oil Palm (Elaeis guineensis Jacq.)

2015 ◽  
Vol 43 (2) ◽  
pp. 147
Author(s):  
Roberdi , ◽  
Sobir , ◽  
Sudirman Yahya ◽  
Nurita Toruan-Mathius ◽  
Tony Liwang
Keyword(s):  
Dna Sequences ◽  
Oil Palm ◽  
Elaeis Guineensis ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Target Sequence ◽  
Polymorphic Markers ◽  
Copia Retrotransposon ◽  
Selection Of

<p>ABSTRACT</p><p>Molecular genetic analysis of hard bunch phenomenon in oil palm was done in order to elucidate the role of genetic factor underlying hard bunch in oil palm plantation. The aim of this study was to identify the AFLP primer combination that co-segregates with hard bunch phenotype related gene in oil palm. Molecular analysis was done by bulk segregant analysis approach. DNA was isolated from leaves of the normal and hard bunch palm. DNA from ten individual palms from each category were pooled and used as a template. A total of 56 AFLP primer combinations were selected for selection of polymorphic primer, and as a result it was found that 22 AFLP primer combinations (39.28%) were polymorphic. A total of 48 individual of palm DNA containing 24 individual for each group were further genotyped by those 22 polymorphic markers. Of these, one AFLP primer combination (E-ACC/M-CTG) was obtained as a co-segregated marker that distinguished the hard bunch DNA from the normal one. Based on the analysis of the target sequence aligned to the oil palm DNA sequences available in database, we found that our sequence has similarity with Ty-1 copia retrotransposon. This sequence distribute in all 16 linkage group of oil palm genome.</p><p>Keywords: abnormal fruits, AFLP, oil palm, Ty-1 copia retrotransposon</p>

scholarly journals Enhanced Cas12a multi-gene regulation using a CRISPR array separator

2021 ◽  
Author(s):  
Jens P. Magnusson ◽  
Antonio R. Rios ◽  
Lingling Wu ◽  
Lei S. Qi
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Gc Content ◽  
Gene Control ◽  
Naturally Occurring ◽  
Crispr Array ◽  
Endogenous Genes ◽  
Simultaneous Activation ◽  
Type V ◽  
Array Performance

AbstractThe type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment a CRISPR separator. Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

scholarly journals Evolutionary dynamics of neutral phenotypes under DNA substitution models

2020 ◽  
Author(s):  
Shadi Zabad ◽  
Alan M Moses
Keyword(s):  
Dna Sequences ◽  
Evolutionary Dynamics ◽  
Gc Content ◽  
Stabilizing Selection ◽  
Restoring Force ◽  
Dna Substitution ◽  
Long Term Trends ◽  
The Mean ◽  
Molecular Phenotypes

AbstractWe study the evolution of quantitative molecular traits in the absence of selection. Using a simple theory based on Felsenstein’s 1981 DNA substitution model, we predict a linear restoring force on the mean of an additive phenotype. Remarkably, the mean dynamics are independent of the effect sizes and genotype and are similar to the widely-used OU model for stabilizing selection. We confirm the predictions empirically using additive molecular phenotypes calculated from ancestral reconstructions of putatively unconstrained DNA sequences in primate genomes. We show that the OU model is favoured by inference software even when applied to GC content of unconstrained sequences or simulations of DNA evolution. We predict and confirm empirically that the dynamics of the variance are more complicated than those predicted by the OU model, and show that our results for the restoring force of mutation hold even for non-additive phenotypes, such as number of transcription factor binding sites, longest encoded peptide and folding propensity of the encoded peptide. Our results have implications for efforts to infer selection based on quantitative phenotype dynamics as well as to understand long-term trends in evolution of quantitative molecular traits.

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