scholarly journals Engineering and optimising deaminase fusions for genome editing

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Luhan Yang ◽  
Adrian W. Briggs ◽  
Wei Leong Chew ◽  
Prashant Mali ◽  
Marc Guell ◽  
...  
Keyword(s):  
Genome Editing ◽  
Binding Sites ◽  
Chromosomal Abnormalities ◽  
Whole Genome ◽  
Bacterial Cells ◽  
Site Specific ◽  
Cytidine Deaminases ◽  
Genome Modification ◽  
Dna Binding Affinity ◽  
Cytidine Deamination

Abstract Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.

scholarly journals Genome Editing With Targeted Deaminases

2016 ◽  
Author(s):  
Luhan Yang ◽  
Adrian W. Briggs ◽  
Wei Leong Chew ◽  
Prashant Mali ◽  
Marc Guell ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zinc Finger Nucleases ◽  
Human Stem Cells ◽  
Site Specific ◽  
Cytidine Deaminases ◽  
Genetic Modifications ◽  
Reduced Toxicity ◽  
A Site ◽  
Dna Template

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

scholarly journals A genome editing primer for the hematologist

Blood ◽  
2016 ◽  
Vol 127 (21) ◽  
pp. 2525-2535 ◽  
Author(s):  
Megan D. Hoban ◽  
Daniel E. Bauer
Keyword(s):  
Genome Editing ◽  
Genetic Alterations ◽  
Double Strand Breaks ◽  
Blood Disorders ◽  
Site Specific ◽  
Strand Breaks ◽  
Epigenome Editing ◽  
Genome Modification ◽  
A Genome ◽  
Experimental Hematology

Abstract Gene editing enables the site-specific modification of the genome. These technologies have rapidly advanced such that they have entered common use in experimental hematology to investigate genetic function. In addition, genome editing is becoming increasingly plausible as a treatment modality to rectify genetic blood disorders and improve cellular therapies. Genome modification typically ensues from site-specific double-strand breaks and may result in a myriad of outcomes. Even single-strand nicks and targeted biochemical modifications that do not permanently alter the DNA sequence (epigenome editing) may be powerful instruments. In this review, we examine the various technologies, describe their advantages and shortcomings for engendering useful genetic alterations, and consider future prospects for genome editing to impact hematology.

scholarly journals Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianhang Wang ◽  
Mingxing Tu ◽  
Ya Wang ◽  
Wuchen Yin ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Editing ◽  
Genome Sequencing ◽  
Plant Biotechnology ◽  
High Specificity ◽  
Fruit Trees ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Indel Mutation ◽  
Target Sites

AbstractThe CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

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 Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

2015 ◽  
Vol 112 (47) ◽  
pp. E6456-E6465 ◽  
Author(s):  
Adrian L. Sanborn ◽  
Suhas S. P. Rao ◽  
Su-Chen Huang ◽  
Neva C. Durand ◽  
Miriam H. Huntley ◽  
...  
Keyword(s):  
Genome Editing ◽  
Binding Sites ◽  
Physical Structure ◽  
Ctcf Binding ◽  
Binding Motifs ◽  
Physical Simulations ◽  
Chromatin Folding ◽  
Mammalian Genomes ◽  
Single Base Pair ◽  
Fractal Globule

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic “tension globule.” In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

scholarly journals Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz0051 ◽  
Author(s):  
Xinyu Ling ◽  
Bingteng Xie ◽  
Xiaoqin Gao ◽  
Liying Chang ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Specific Chemical ◽  
Site Specific ◽  
Additional Tool ◽  
Homology Directed Repair ◽  
Chemically Modified ◽  
Dna Template ◽  
Oligonucleotide Conjugates

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

scholarly journals LmbU directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780, which are located outside the lmb cluster and inhibit lincomycin biosynthesis in Streptomyces lincolnensis

2021 ◽  
Author(s):  
Bingbing Hou ◽  
Xianyan Zhang ◽  
Yue Mao ◽  
Ruida Wang ◽  
Jiang Ye ◽  
...  
Keyword(s):  
Binding Sites ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Whole Genome ◽  
Biosynthetic Gene ◽  
Qrt Pcr ◽  
Dna Binding Sites ◽  
Reporter Assays ◽  
Streptomyces Lincolnensis

The productions of antibiotics are usually regulated by cluster-situated regulators (CSRs), which can directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). However, few studies have looked into the regulation of CSRs on the targets outside the BGC. Here, we screened the targets of LmbU in the whole genome of S. lincolnensis, and found 14 candidate targets, among of which, 8 targets can bind to LmbU by EMSAs. Reporter assays in vivo revealed that LmbU repressed transcription of SLINC_RS02575 and SLINC_RS05540, while activated transcription of SLINC_RS42780. In addition, disruptions of SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 promoted the production of lincomycin, and qRT-PCR showed that SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. What's more, the homologues of LmbU and its targets SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 are widely found in actinomycetes, while the distributions of DNA-binding sites (DBS) of LmbU are diverse, indicating the regulatory mechanisms of LmbU homologues in various strains are different and complicated.

scholarly journals Whole Genome Sequence Analysis and Homology Modelling of a 3C Like Peptidase and a Non-Structural Protein 3 of the SARS-CoV-2 Shows Protein Ligand Interaction with an Aza-Peptide and a Noncovalent Lead Inhibitor with Possible Antiviral Properties

2020 ◽  
Author(s):  
Arun Shanker ◽  
Divya Bhanu ◽  
Anjani Alluri
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Tertiary Structure ◽  
Vaccine Development ◽  
Homology Modelling ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Structural Protein ◽  
Ligand Interaction ◽  
Percent Identity

<p></p><p>The family of viruses belonging to Coronaviridae mainly consist of virulent pathogens that have a zoonotic property, Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS-CoV) of this family have emerged before and now the SARS-CoV-2 has emerged in China. Characterization of spike glycoproteins, polyproteins and other viral proteins from viruses are important for vaccine development. Homology modelling of these proteins with known templates offers the opportunity to discover ligand binding sites and explore the possible antiviral properties of these protein ligand complexes. Any information emerging from these protein models can be used for vaccine development. In this study we did a complete bioinformatic analysis, sequence alignment, comparison of multiple sequences and homology modelling of the <a>SARS-CoV-2 </a>whole genome sequences, the spike protein and the polyproteins for homology with known proteins, we also analysed receptor binding sites in these models for possible binding with ligands that exhibit antiviral properties. Our results showed that the tertiary structure of the polyprotein isolate SARS-CoV-2_HKU-SZ-001_2020 had 98.94 percent identity with SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors. <a>Our results indicate that a part of the viral genome </a><a>(residues 3268 -3573 in Frame 2 with 306 amino acids) of the SARS-CoV-2 virus isolate Wuhan-Hu-1 (Genbank Accession Number MN908947.3) </a>when modelled with template 2a5i of the PDB database had 96 percent identity with a 3C like peptidase of SARS-CoV which has ability to bind with Aza-Peptide Epoxide (APE) which is known for irreversible inhibition of SARS-CoV main peptidase. The part of the genome (residues 1568-1882 in Frame 2 with 315 amino acids) when modelled with template 3e9s of the PDB database had 82 percent identity with a papain-like protease/deubiquitinase which when complexed with ligand GRL0617 acts as inhibitor which can block SARS-CoV replication. The regions studied was conserved in more than 90 genomes of SARS-CoV-2. It is possible that these viral inhibiters can be used for vaccine development for the SARS-CoV-2.</p><p></p><p></p>

scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

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