To target rat Piezo1, we searched for protospacer adjacent motif (PAM) sequences (NGG for Streptococcus pyogenes Cas9) within the protein coding sequences of rat Piezo1 and designed 13 different single guide RNAs (sgRNAs) to target this region (Table S1, Supporting Information).The activities of these sgRNAs were screened after transfection of sgRNA‐Cas9 RNP complexes into a rat C6 glioma cell line and selected six sequences that demonstrated an indel frequency greater than 90% using targeted deep sequencing (Figure S13A, Supporting Information). This evidence concerns the gene RNPC3 and central nervous system cancer.