The occurrence of random mutations can increase the diversity of the genome and promote the evolutionary process of organisms. High efficiency mutagenesis techniques significantly accelerate the evolutionary process. In this work, we describe a targeted in vivo mutagenesis system to significantly increase mutation frequency and generate mutations across all four nucleotides. We constructed different DNA-modifying enzyme-PmCDA1-T7 RNA polymerase fusion proteins, achieved targeted mutagenesis by flanking the target gene with T7 promoters, and tuned the mutation spectra by introducing different DNA-modifying enzymes. With the mutagenesis fusion proteins, the mutation frequency of the target gene could reach 5.13x10-3, and the proportion of non-C→T mutations is 10~11-fold higher than the cytidine-based evolutionary tools. We also demonstrated that our mutagenesis tools could be used to evolve the essential enzyme in the β-carotene biosynthesis process and generate mutations with different types.