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In <i>Arabidopsis,</i> the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) functions as a main regulator of the cold signaling. In this study, CRISPR/Cas9-mediated targeted mutagenesis of the <i>HOS1</i> gene in the first exon was performed. DNA sequencing showed that frameshift indels introduced by genome editing of <i>HOS1</i> resulted in the appearance of premature stop codons, disrupting the open reading frame. Obtained <i>hos1</i>^Cas9 mutant plants were compared with the SALK T-DNA insertion mutant, line <i>hos1-3</i>, in terms of their tolerance to abiotic stresses, accumulation of secondary metabolites and expression levels of genes participating in these processes. Upon exposure to cold stress, enhanced tolerance and expression of cold-responsive genes were observed in both <i>hos1-3</i> and <i>hos1</i>^Cas9 plants. The <i>hos1</i> mutation caused changes in the synthesis of phytoalexins in transformed cells. The content of glucosinolates (GSLs) was down-regulated by 1.5-times, while flavonol glycosides were up-regulated by 1.2 to 4.2 times in transgenic plants. The transcript abundance of the corresponding MYB and bHLH transcription factors, which are responsible for the regulation of secondary metabolism in <i>Arabidopsis,</i> were also altered. Our data suggest a relationship between HOS1-regulated downstream signaling and phytoalexin biosynthesis.