Isoform-specific CRISPR knockout of Trp73 in mice

The CRISPR/Cas9 system was utilized for isoform-specific knockout of the Trp73 gene in C57BL/6J mice. Two types of small RNAs, target-recognizing CRISPR RNA (crRNA) designed with CHOPCHOP (48 (link)), and auxiliary transactivating crRNA (tracrRNA), were purchased from FASMAC as listed in Supplementary Table S3. We delivered TrueCut Cas9 Protein v2 (Thermo Fisher Scientific) together with the crRNA and tracrRNA into in vitro–fertilized eggs of C57BL/6J mice according to the institutional procedure of Kyoto University (49 (link)). Both TAp73 and DNp73 knockout genotypes were obtained with 1bp insertions on the crRNA recognizing region, which results in truncating (frameshift) mutations (Supplementary Fig. S4A). Genotyping for each knockout isoform was performed with restriction enzyme digestion of PCR-amplified DNA. For TAp73, genomic DNA was amplified with TAp73-genotype-F and TAp73-genotype-R primers (Supplementary Table S3), followed by BstBI (New England Biolabs) digestion, whereas DNp73-genotype-F and DNp73-genotype-R primers (Supplementary Table S3), and MseI (New England Biolabs) were similarly utilized for DNp73 genotyping.

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Toyoda K., Yasunaga J.I., Shichijo T., Arima Y., Tsujita K., Tanaka A., Salah T., Zhang W., Hussein O., Sonoda M., Watanabe M., Kurita D., Nakashima K., Yamada K., Miyoshi H., Ohshima K, & Matsuoka M. (2023). HTLV-1 bZIP Factor-Induced Reprogramming of Lactate Metabolism and Epigenetic Status Promote Leukemic Cell Expansion. Blood Cancer Discovery, 4(5), 374-393.

Publication 2023

TrueCut Cas9 Protein v2 tracrRNA BstBI

Corresponding Organization :

Other organizations : Kumamoto University, The University of Tokyo, Kurume University

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Variable analysis

independent variables

Utilization of the CRISPR/Cas9 system for isoform-specific knockout of the Trp73 gene in C57BL/6J mice

dependent variables

Obtaining both TAp73 and DNp73 knockout genotypes with 1bp insertions on the crRNA recognizing region, which results in truncating (frameshift) mutations
Genotyping for each knockout isoform performed with restriction enzyme digestion of PCR-amplified DNA

control variables

C57BL/6J mice used as the experimental model
TrueCut Cas9 Protein v2 (Thermo Fisher Scientific) delivered together with the crRNA and tracrRNA into in vitro–fertilized eggs of C57BL/6J mice according to the institutional procedure of Kyoto University

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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