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THE USE OF CRISPR-CAS9 TECHNOLOGY TO KNOCK OUT GENES ENCODING INTRACELLULAR PROTEINS WITH LOW EXPRESSION (EXEMPLIFIED BY NOD1 AND NOD2)


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Abstract

The CRISPR-Cas9 technology allows to rapidly knock out genes in eukaryotic cells. One of the main problems with the use of CRISPR-Cas9 is the selection of viable cells carrying functionally significant mutations of the target gene. This problem is especially evident when the protein encoded by the target gene is expressed intracellularly at a low level that cannot be detected by routine techniques. In these cases, the criterion for the selection of knock-out cells could be lack of function of the target protein, provided that this function is known and can be measured. In such situations, the experimental technique consists of following steps: (1) the cells are transfected with a reporter construct that allows to measure function of the target gene; 2) the target gene is knocked-out using Cas9 nickase; (3) single-cell clones are obtained and screened for target gene function using the reporter construct introduced earlier; (4) non-responding clones are selected; (5) the selected clones are tested again for the function of target gene and absence of significant off-target effects; (6) knock-out of the target gene is verified by genetic analysis. The successful use of this technique is illustrated by knocking out NOD1 and NOD2 genes, which code receptors to peptidoglycan fragments, in HEK293T cells.


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