Genome editing has revolutionized speed breeding by enabling researchers to alter the genome directly as desired. The most widely used CRISPR/Cas9 technology has mainly been applied to engineer null mutations in coding sequences for the creation of loss-of-function alleles. Upstream regulatory elements of the promoter region and 5′ upstream open reading frame (uORF) have been engineered to generate gain-of-function alleles. Nonetheless, more approaches for gene knock-up to enhance endogenous plant protein levels without affecting the coding region are needed for crop breeding. Recent studies have demonstrated that inhibitory regions within the 3′ UTRs can reduce protein production by interfering with translation. We hypothesized that CRISPR/Cas9-mediated deletion of inhibitory regions in the 3′ UTRs might provide an efficient method for generating artificial alleles for enhanced protein levels and desired plant traits. Thus, we chose two rice genes OsLTT7 (low temperature tolerance 7) and OsSBI (shortened basal internodes), and an Arabidopsis gene AtTPPD (trehalose-6-phosphate phosphatase D) for the test. We first identified the inhibitory regions within the target 3′ UTRs using a dual-luciferase reporter system in plant protoplasts. Then, we used CRISPR/Cas9 technology to edit the in vivo inhibitory regions in the target 3′ UTRs. We ultimately obtained two homozygous transgene-free T₂ targeted deletion lines for each target gene. We found that levels of the targeted proteins were consistently increased in these edited lines, the OsLTT7^3′utr increased cold tolerance, the OsSBI^3′utr exhibited a dwarf phenotype, and the AtTPPD^3′utr increased salt tolerance. Thus, this study provides a transgene-free CRISPR/Cas9-mediated approach for gene knock-up that may serve as a breeding strategy in different crop species.