Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

Lee, Jae Seong; Kallehauge, Thomas Beuchert; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup

doi:10.1038/srep08572

Cited by 182 publications

(201 citation statements)

References 38 publications

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“…S2c). To demonstrate the true potential of this site‐specific integration, we isolated single cell clones with confirmed integration into the loci using a PCR‐based approach . In this case, site‐specific integration and isolation demonstrated a 3.1‐ to 3.9‐fold improvement in geometric mean fluorescence (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identifying and retargeting transcriptional hot spots in the human genome

Cheng

Lewis

Kim

et al. 2016

Biotechnology Journal

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Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site-specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single-integrant, recombinant human cell lines that exhibit stable, high-level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non-protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12(th) exon-intron region of the GRIK1 gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4-fold. The information identified here along with recent advances in site-specific genomic editing techniques can lead to expedited cell line development.

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Section: Resultsmentioning

confidence: 99%

Identifying and retargeting transcriptional hot spots in the human genome

Cheng

Lewis

Kim

et al. 2016

Biotechnology Journal

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“…After propagation of yeast transformants in liquid media, the DNA from whole cultures was extracted using ZR Fungal/Bacterial DNA miniprep kit (Zymo Research). Both mutagenized fragments and genomic DNA were used to generate amplicons of BTS1 and HMG2 as described by Lee and colleagues (Lee et al, 2015) with the following primers: TJOS-339F and TJOS-339R; TJOS-340F and TJOS-340R respectively. Amplicons were sequenced by Illumina MiSeq using Nextera DNA library prep kit (Illumina).…”

Section: Sequencing and Analysismentioning

confidence: 99%

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

Jakočiūnas

Pedersen

Lis

et al. 2018

Metabolic Engineering

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“…In addition, the use of a stable cell clone has an advantage in terms of the high reproducibility of the quality and quantity of the expression products compared to transient‐expression. In this study, a stable cell construction was achieved by a classical transfection and selection method, but the process is expected to be improved by the use of recently developed genome editing techniques, such as the CRISPR/Cas9 system . One drawback of the stable expression compared to the transient expression is that the former requires more time to obtain the purification results from the system.…”

Section: Discussionmentioning

confidence: 99%

A large‐scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand‐binding domains of taste receptor

2017

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Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.

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Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

Cited by 182 publications

References 38 publications

Identifying and retargeting transcriptional hot spots in the human genome

Identifying and retargeting transcriptional hot spots in the human genome

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

A large‐scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand‐binding domains of taste receptor

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