CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.