Selecting for CRISPR-Edited Knock-In Cells

Reuven, Nina; Shaul, Yosef

doi:10.3390/ijms231911919

Cited by 5 publications

(4 citation statements)

References 76 publications

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“…Since we performed transient transfection using a lipid-based reagent, no stable BRCA-depleted colony was generated. To circumvent this problem, co-delivery of a CRISPR/Cas9 RNP complex and a plasmid containing a selectable marker could enrich for edited cells 41 . Moreover, plasmid-based knockout strategies can also generate stable cell lines, particularly with lentiviral transduction.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines

Witz,

Dardare,

Francois

et al. 2023

Sci Rep

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Pancreatic cancer is one of the most aggressive diseases with a very poor outcome. Olaparib, a PARP inhibitor, as maintenance therapy showed benefits in patients with metastatic pancreatic adenocarcinoma bearing germline BRCA1/2 mutations. However, germline BRCA mutation has been described in only 4–7% of patients with pancreatic adenocarcinoma. A CRISPR/Cas9-mediated system was used to knock-in the c.763G > T p.(Glu255*) and c.2133C > A p.(Cys711*) mutations in cell lines to obtain truncated BRCA1 and BRCA2 proteins, respectively. A CRISPR/Cas9 ribonucleoprotein complex was assembled for each mutation and transfected into two pancreatic cell lines (T3M4 and Capan-2) and into a breast cancer cell lines (MCF7) as control. BRCA protein levels were significantly decreased in all BRCA-depleted cells (P < 0.05), proving the transfection efficiency of our CRISPR/Cas9 systems. As expected, the calculated olaparib IC50 were significantly reduced for all cell lines harbored BRCA1 or BRCA2 mutations compared to wild-type BRCA1/2 cells (P < 0.01). Furthermore, we observed a higher induction of apoptosis after 72 h olaparib treatment in BRCA-depleted cells than in wild-type cells. This strategy might offer new insights into the management of patients with pancreatic cancer and open up new perspectives based on the in vivo use of CRISPR/Cas9 strategy.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines

Witz,

Dardare,

Francois

et al. 2023

Sci Rep

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“…The efficiency of simultaneous editing of all genes is equal to the product of the efficiency of editing all individual genes [ 136 ]. If individual genes are edited too inefficiently, it makes later screening and identification doubly difficult and time-consuming, so it is also important not to express too many gRNAs when constructing editing vectors [ 137 ]. Although it is difficult to obtain higher-order mutants using a single editing vector, we can use crosses between stably inherited mutants of different genes to obtain multiple mutants [ 138 , 139 , 140 ].…”

Section: Strategies and Methods For Optimizing Crispr/cas9 Gene-editi...mentioning

confidence: 99%

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Zhou

Luan

Liu

et al. 2023

Plants

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Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

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“…CRISPR/Cas has democratized the generation of genetically engineered cell lines for studies of genotype–phenotype relationships, but generating a clonal cell line with the desired genotype may require labor-intensive screening of hundreds of clones to identify a correct one. Enrichment may facilitate the direct identification and isolation of very infrequent genotypes in a population or at least vastly increase the likelihood of identifying a correct clone, thereby reducing the labor intensity [ 57 , 58 ].…”

Section: Selection Of Gene Edited Cellsmentioning

confidence: 99%

Enrichment strategies to enhance genome editing

Mikkelsen

Bak

2023

J Biomed Sci

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Genome editing technologies hold great promise for numerous applications including the understanding of cellular and disease mechanisms and the development of gene and cellular therapies. Achieving high editing frequencies is critical to these research areas and to achieve the overall goal of being able to manipulate any target with any desired genetic outcome. However, gene editing technologies sometimes suffer from low editing efficiencies due to several challenges. This is often the case for emerging gene editing technologies, which require assistance for translation into broader applications. Enrichment strategies can support this goal by selecting gene edited cells from non-edited cells. In this review, we elucidate the different enrichment strategies, their many applications in non-clinical and clinical settings, and the remaining need for novel strategies to further improve genome research and gene and cellular therapy studies.

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Selecting for CRISPR-Edited Knock-In Cells

Cited by 5 publications

References 76 publications

CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines

CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Enrichment strategies to enhance genome editing

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