scholarly journals DNA templates with blocked long 3' end single-stranded overhangs (BL3SSO) promote bona fide Cas9-stimulated homology-directed repair of long transgenes into endogenous gene loci

2021 ◽  
Author(s):  
Saptaparni Bandyopadhyay ◽  
Joseph Douglass ◽  
Sebastian Kapell ◽  
Nazimuddin Khan ◽  
Fabiana Feitosa-Suntheimer ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Hek293 Cell ◽  
Biological Processes ◽  
Human Cell Lines ◽  
Dna Templates ◽  
Endogenous Gene ◽  
Homology Directed Repair ◽  
Bona Fide ◽  
Endogenous Loci ◽  
Plasmid Backbone

Abstract Knock-in of large transgenes by Cas9-mediated homology-directed repair (HDR) is an extremely inefficient process. Although the use of single-stranded oligonucleotides (ssODN) as an HDR donor has improved the integration of smaller transgenes, they do not support efficient insertion of large DNA sequences. In an effort to gain insights into the mechanism(s) governing the HDR-mediated integration of larger transgenes and to improve the technology, we conducted knock-in experiments targeting the human EMX1 locus and applied rigorous genomic PCR analyses in the human HEK293 cell line. This exercise revealed an unexpected molecular complication arising from the transgene HDR being initiated at the single homology arm and the subsequent genomic integration of plasmid backbone sequences. To pivot around this problem, we devised a novel PCR-constructed template containing Blocked Long 3' Single-Stranded Overhangs (BL3SSO) that greatly improved the efficiency of bona fide Cas9-stimulated HDR at the EMX1 locus. We further refined BL3SSO technology and successfully used it to insert GFP transgenes into two important interferon-stimulated gene (ISG) loci, Viperin/RSAD2 and ISG15. This study demonstrates the utility of the BL3SSO platform for inserting long DNA sequences into both constitutive and inducible endogenous loci to generate novel human cell lines for the study of important biological processes.

scholarly journals SiteOut: an online tool to design binding site-free DNA sequences

2015 ◽  
Author(s):  
Javier Estrada ◽  
Teresa Ruiz-Herrero ◽  
Clarissa Scholes ◽  
Zeba Wunderlich ◽  
Angela DePace
Keyword(s):  
Binding Site ◽  
Dna Sequences ◽  
Binding Sites ◽  
Regulatory Proteins ◽  
Biological Processes ◽  
Major Goal ◽  
Specific Sequence ◽  
Online Tool ◽  
Protein Binding Sites ◽  
Regulatory Dna

DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/

scholarly journals Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

2020 ◽  
Vol 6 (7) ◽  
pp. eaax2941 ◽  
Author(s):  
Boris V. Skryabin ◽  
Delf-Magnus Kummerfeld ◽  
Leonid Gubar ◽  
Birte Seeger ◽  
Helena Kaiser ◽  
...  
Keyword(s):  
Conditional Knockout ◽  
Nonhomologous End Joining ◽  
High Rate ◽  
Model Organisms ◽  
Pcr Analysis ◽  
End Joining ◽  
Dna Templates ◽  
Homology Directed Repair ◽  
Conditional Knockout Mouse ◽  
Wide Range

CRISPR-Cas9–mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.

scholarly journals Targeted Gene Editing in Plants using CRISPR-Cas9, Single-Stranded DNA Oligonucleotides, and Protoplast Regeneration

2021 ◽  
Author(s):  
Chen-Tran Hsu ◽  
Yu-Hsuan Yuan ◽  
Yao-Cheng Lin ◽  
Steven Lin ◽  
Qiao-Wei Cheng ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Nonhomologous End Joining ◽  
Protoplast Regeneration ◽  
End Joining ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Dna Oligonucleotides ◽  
Vector Construction ◽  
Regenerated Plants ◽  
Low Efficiency

AbstractGenome editing requires insertion of DNA sequences into specific locations. Protocols involving clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins rely on homology-directed repair, require laborious vector construction, and have low efficiency. DNA oligonucleotides can be used as donors for targeted insertion via nonhomologous end joining. Our simple protocol eliminates the need for expensive equipment and vector construction by using polyethylene glycol to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. We achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated plants showed targeted insertions, and 1 contained a precise insertion of a 6-bp EcoRI site. Whole-genome sequencing showed that the DNA inserted only in the targeted positions, and genetic analysis showed that the inserted sequences transmitted to the next generation.

scholarly journals MiPepid: MicroPeptide identification tool using machine learning

2019 ◽  
Author(s):  
Mengmeng Zhu ◽  
Michael Gribskov
Keyword(s):  
Machine Learning ◽  
Dna Sequences ◽  
Biological Activities ◽  
Prediction Methods ◽  
Sequence Information ◽  
Blind Test ◽  
Small Proteins ◽  
Bona Fide ◽  
Genome Scale ◽  
Coding Potential

Abstract Background Micropeptides are small proteins with a length <= 100 amino acids. They were traditionally ignored as few were discovered due to technical difficulties. In the past decade, a growing number of micropeptides have been shown to play significant roles in vital biological activities. Despite the increased amount of data, we still lack bioinformatics tools specifically for identifying micropeptides from DNA sequences. Indeed, most existing tools for classifying coding and noncoding ORFs were built on datasets in which “normal-sized” proteins are considered to be positives and short ORFs are generally considered to be noncoding. Since the functional and biophysical constraints on small peptides are likely to be different from those on “normal” proteins, methods for predicting short translated ORFs must be trained independently from those for longer proteins. Results In this study, we developed MiPepid, a machine-learning tool specifically for the identification of micropeptides. We trained MiPepid using carefully cleaned data from existing databases and logistic regression with 4-mer features. With only the sequence information of an ORF, MiPepid is able to predict whether it encodes a micropeptide with 96% accuracy on a blind dataset of high-confidence micropeptides, and to correctly classify newly discovered micropeptides not included in either the training or the blind test data. Compared with state-of-the-art coding potential prediction methods, MiPepid performs exceptionally well, as other methods incorrectly classify most bona fide micropeptides as noncoding. MiPepid is alignment-free and runs sufficiently fast for genome-scale analyses. It is easy to use and is available at https://github.com/MindAI/MiPepid. Conclusion MiPepid was developed to specifically predict micropeptides, a category of proteins with increasing significance, from DNA sequences. It shows evident advantages over existing coding potential prediction methods on micropeptide identification. It is ready to use and runs fast.

scholarly journals Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

2020 ◽  
Author(s):  
Joseph R. Owen ◽  
Sadie L. Hennig ◽  
Bret R. McNabb ◽  
Jason C. Lin ◽  
Amy E. Young ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Bovine Genome ◽  
Direct Injection ◽  
End Joining ◽  
Homology Directed Repair ◽  
Livestock Breeding ◽  
Repair Mechanisms ◽  
Dividing Cells ◽  
The One ◽  
Donor Template

ABSTRACTIntroducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology-directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency is low in zygotes, especially for the introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor template, or a HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

scholarly journals Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

2021 ◽  
Author(s):  
Sophie Hertel ◽  
Richard Spinney ◽  
Stephanie Xu ◽  
Thomas E Ouldridge ◽  
Richard Morris ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Dna Sequences ◽  
Physical Factors ◽  
Biological Processes ◽  
Physical Mechanisms ◽  
Mechanistic Insight ◽  
Sequence Dependent ◽  
Dna Hybridisation ◽  
Kinetics Of ◽  
Insight Into

The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridise at different rates are not well understood. Secondary structure is one predictable factor that influences hybridisation rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. Consequently, to achieve a good correlation with experimental data, current prediction algorithms require many parameters that provide little mechanistic insight into DNA hybridisation. In this context, we measured hybridisation rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify their hybridisation rates. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters, thus providing new insight into the physical factors underpinning DNA hybridisation rates.

scholarly journals Visualizing the protons in a metalloenzyme electron proton transfer pathway

2020 ◽  
Vol 117 (12) ◽  
pp. 6484-6490 ◽  
Author(s):  
Hanna Kwon ◽  
Jaswir Basran ◽  
Juliette M. Devos ◽  
Reynier Suardíaz ◽  
Marc W. van der Kamp ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Transfer ◽  
Hydrogen Bonding ◽  
Proton Transfer ◽  
Proton Donor ◽  
Biological Processes ◽  
Proton Coupled Electron Transfer ◽  
Neutron Structure ◽  
Bona Fide ◽  
Electron Proton Transfer

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.

scholarly journals DNA polymerase activity on synthetic N3′→P5′ phosphoramidate DNA templates

2019 ◽  
Vol 47 (17) ◽  
pp. 8941-8949 ◽  
Author(s):  
Victor S Lelyveld ◽  
Derek K O’Flaherty ◽  
Lijun Zhou ◽  
Enver Cagri Izgu ◽  
Jack W Szostak
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Chemical Space ◽  
Genetic Material ◽  
Polymerase Activity ◽  
Dna Duplexes ◽  
Dna Templates ◽  
Reverse Transcriptases ◽  
The Universe ◽  
Rna And Dna

Abstract Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3′→P5′ phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.

scholarly journals Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

2020 ◽  
Vol 48 (14) ◽  
pp. 8178-8187 ◽  
Author(s):  
Ivana Indikova ◽  
Stanislav Indik
Keyword(s):  
Genome Editing ◽  
High Throughput Sequencing ◽  
Target Cells ◽  
Egfp Gene ◽  
Endogenous Gene ◽  
Highly Efficient ◽  
Transgene Delivery ◽  
Responsive Element ◽  
Endogenous Loci ◽  
Spatiotemporal Control

Abstract The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to &gt;98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.

scholarly journals A novel role of MNT as a negative regulator of REL and the NF-κB pathway

Oncogenesis ◽  
2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Judit Liaño-Pons ◽  
M. Carmen Lafita-Navarro ◽  
Lorena García-Gaipo ◽  
Carlota Colomer ◽  
Javier Rodríguez ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Transcriptional Regulator ◽  
Negative Regulator ◽  
Biological Processes ◽  
Helix Loop Helix ◽  
Bona Fide ◽  
E Boxes ◽  
Independent Protein

AbstractMNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix–loop–helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT–REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT–REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

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