A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles

Joberty, Gérard; Fälth-Savitski, Maria; Paulmann, Marcel; Bösche, Markus; Doce, Carola; Cheng, Aaron T.; Drewes, Gerard; Grandi, Paola

doi:10.1089/crispr.2019.0064

Cited by 12 publications

(13 citation statements)

References 35 publications

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“…A recent approach developed to bypass clonal isolation after CRISPR‐Cas9 editing in CHO cells may offer a viable solution in this regard. [ 52 ] Overall, this study demonstrates the utility of CRISPR‐Cas9 for generating gene‐knockout Sf9 cell lines with novel phenotypes relevant to the industrial production of BEVS‐derived biologics.…”

Section: Discussionmentioning

confidence: 84%

Knockout of Sf‐Caspase‐1 generates apoptosis‐resistant Sf9 cell lines: Implications for baculovirus expression

Malmanche

Marcellin

Reid

2022

Biotechnology Journal

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The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing βgalactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.

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Section: Discussionmentioning

confidence: 84%

Knockout of Sf‐Caspase‐1 generates apoptosis‐resistant Sf9 cell lines: Implications for baculovirus expression

Malmanche

Marcellin

Reid

2022

Biotechnology Journal

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“…Although the use of one gRNA is frequently sufficient to knock out the target gene, exon skipping and alternative splicing may occur, resulting in the production of fully or partially functional proteins, and allowing the cells to bypass single Cas9‐induced double-strand breaks ( 43 ). To maximize the likelihood of knocking out a target gene, a two-guide strategy has been described ( 44 ). In this study, we used dual gRNAs targeting two distinct sites to introduce double cuts which resulted in the elimination of the DNA between the two targeted sites as confirmed by PCR and Sanger sequencing.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells

et al. 2021

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Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout in the human hepatoblastoma cell line, HuH6. PIM3 knockout of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 knockout downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 knockout decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 knockout cells rescued the malignant phenotype. Successful CRISPR/Cas9 knockout of PIM3 kinase in human hepatoblastoma cells confirmed the role of PIM3 in promoting hepatoblastoma tumorigenesis and cancer cell stemness.

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“…В работе [10] показано, что при двойном нокауте sgRNA демонстрируют синергетический эффект, если сайты Cas9 располагаются на расстоянии 40 -300 пар нуклеотидов (п. н.)…”

Section: результаты и их обсуждениеunclassified

“…друг от друга. Также было отмечено, что синергетический эффект отсутствует, если расстояние между сайтами Cas9 двух sgRNA составляет менее 35 п. н., при этом эффективность редактирования может быть ниже эффективности редактирования при использовании данных sgRNA по отдельности [10].…”

Section: результаты и их обсуждениеunclassified

Knockout of the T-cell receptor and HLA class I genes in human cells using the CRISPR /Cas9 system

Kushniarova¹,

Migas²,

Klych³

et al. 2022

Experimental Biology and Biotechnology

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The CRISPR /Cas9 system has found a wide application in cell biology as a tool for gene knockout. In particular, the CRISPR /Cas9 system is used to create allogeneic CAR-T lymphocytes by knocking out the genes TRAC, TRBC1, TRBC2 and B2M. To obtain a large number of cells of the desired phenotype, it is necessary to optimise the process of genomic editing, the effectiveness of which is determined by the sgRNA used. In this work, we experimentally determined the most effective sequences that allow to obtain up to 60.3 % of cells negative for the expression of the B2M protein and up to 71.8 % of cells negative for the expression of the T-cell receptor. It has also been shown that the simultaneous use of two sgRNAs for gene knockout demonstrates a lower efficiency compared to using these sgRNAs separately.

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A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles

Cited by 12 publications

References 35 publications

Knockout of Sf‐Caspase‐1 generates apoptosis‐resistant Sf9 cell lines: Implications for baculovirus expression

Knockout of Sf‐Caspase‐1 generates apoptosis‐resistant Sf9 cell lines: Implications for baculovirus expression

CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells

Knockout of the T-cell receptor and HLA class I genes in human cells using the CRISPR /Cas9 system

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