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 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.

DNA synthesis errors associated with double-strand-break repair.

Genetics ◽  
1995 ◽  
Vol 140 (3) ◽  
pp. 965-972 ◽  
Author(s):  
J N Strathern ◽  
B K Shafer ◽  
C B McGill
Keyword(s):  
Dna Synthesis ◽  
Dna Cleavage ◽  
Genome Duplication ◽  
Inducible Promoter ◽  
Strand Break ◽  
Site Specific ◽  
Large Populations ◽  
Dna Break ◽  
A Site ◽  
Ho Gene

Abstract Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

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 Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

2017 ◽  
Vol 13 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Danielle N. Gallagher ◽  
James E. Haber
Keyword(s):  
Dna Cleavage ◽  
Gene Editing ◽  
Site Specific ◽  
A Site

scholarly journals Application of genome-editing systems to enhance available pig resources for agriculture and biomedicine

2020 ◽  
Vol 32 (2) ◽  
pp. 40
Author(s):  
Kiho Lee ◽  
Kayla Farrell ◽  
Kyungjun Uh
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Direct Injection ◽  
Somatic Cells ◽  
Cost Effective ◽  
Genetically Engineered ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Developmental Defects ◽  
Site Specific

Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.

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