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A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells

Published in DNA Repair3.71
· DOI :10.1016/j.dnarep.2019.102691
Sandra Bosshard2
Estimated H-index: 2
(UNIL: University of Lausanne),
Pierre-Olivier Duroy (UNIL: University of Lausanne), Nicolas Mermod30
Estimated H-index: 30
(UNIL: University of Lausanne)
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Abstract
Abstract CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR. This dependency was influenced by the CRISPR nuclease type and the DSB-to-mutation distance, but not by the DNA sequence surrounding the DSBs or reporter cell line. We also identified elevated Mre11 and Pari, and low Rad51 expression levels as the most rate-limiting factors for HR in CHO cells. Counteracting these three bottlenecks improved precise genome editing by up to 75%. Altogether, our study provides novel insights into the complex interplay of alt-EJ and HR repair pathways, highlighting their relevance for developing improved genome editing strategies.
  • References (86)
  • Citations (0)
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References86
Newest
Published in Nature Communications11.88
Stephan Riesenberg2
Estimated H-index: 2
(MPG: Max Planck Society),
Tomislav Maricic15
Estimated H-index: 15
(MPG: Max Planck Society)
A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks which are then repaired by homology-directed repair using DNA donor molecules carrying desired mutations. Using a mixture of small molecules, the “CRISPY” mix, we achieve a 2.8- to 7.2-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes...
Alexanda K. Ling3
Estimated H-index: 3
(U of T: University of Toronto),
Clare C. So3
Estimated H-index: 3
(U of T: University of Toronto)
+ 3 AuthorsAlberto Martin24
Estimated H-index: 24
(U of T: University of Toronto)
Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greate...
Brenda Lemos3
Estimated H-index: 3
(Brandeis University),
Adam C. Kaplan1
Estimated H-index: 1
(Brandeis University)
+ 5 AuthorsJames E. Haber89
Estimated H-index: 89
(Brandeis University)
Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-...
Published on Dec 15, 2017in Nucleic Acids Research11.15
Varandt Y. Khodaverdian2
Estimated H-index: 2
(Tufts University),
Terrence Hanscom1
Estimated H-index: 1
(Tufts University)
+ 5 AuthorsMitch McVey17
Estimated H-index: 17
(Tufts University)
Published on Dec 1, 2017in Nature Communications11.88
Alexander Zelensky4
Estimated H-index: 4
(EUR: Erasmus University Rotterdam),
Joost Schimmel1
Estimated H-index: 1
(LEI: Leiden University)
+ 2 AuthorsMarcel Tijsterman30
Estimated H-index: 30
(LEI: Leiden University)
Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events,...
Published on Dec 1, 2017in Nature Communications11.88
Shinta Saito4
Estimated H-index: 4
,
Ryo Maeda4
Estimated H-index: 4
,
Noritaka Adachi29
Estimated H-index: 29
Homologous recombination mediated gene targeting is highly inefficient in human cells due to random integration events, Here the authors show that dual repression of polymerase θ and DNA ligase IV eliminate random integration events.
Published on Dec 1, 2017in Nature Communications11.88
Jared Carlson-Stevermer5
Estimated H-index: 5
(UW: University of Wisconsin-Madison),
Amr A. Abdeen11
Estimated H-index: 11
(UW: University of Wisconsin-Madison)
+ 4 AuthorsKrishanu Saha21
Estimated H-index: 21
(UW: University of Wisconsin-Madison)
Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edite...
Published on Aug 1, 2017in Nature43.07
Hong Ma27
Estimated H-index: 27
(Oregon National Primate Research Center),
Nuria Marti-Gutierrez1
Estimated H-index: 1
(OHSU: Oregon Health & Science University)
+ 28 AuthorsRiffat Ahmed10
Estimated H-index: 10
(OHSU: Oregon Health & Science University)
CRISPR–Cas9 genome editing is used to induce a DNA repair response and correct a disease-causing heterozygous mutation in human embryos with reduced mosaicism and preferential repair using the wild-type copy of the gene.
Published on Jun 1, 2017in Nature Methods28.47
D. Agudelo15
Estimated H-index: 15
,
Alexis Duringer1
Estimated H-index: 1
+ 9 AuthorsGraham Dellaire35
Estimated H-index: 35
A gain-of-function mutation in a sodium/potassium pump renders cells resistant to a small-molecule drug and provides an efficient coselection strategy to enrich for CRISPR-induced mutations at an independent locus.
Published on Feb 1, 2017in Biotechnology and Bioengineering4.26
Kaja Kostyrko5
Estimated H-index: 5
(UNIL: University of Lausanne),
Samuel Neuenschwander11
Estimated H-index: 11
(Swiss Institute of Bioinformatics)
+ 9 AuthorsPierre-Alain Girod14
Estimated H-index: 14
Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest...
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