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. 2022 May-Jun;24(3):266-272.
doi: 10.4103/aja.aja_63_21.

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice

Yuki Oyama  1   2 , Haruhiko Miyata  2 , Keisuke Shimada  2 , Yoshitaka Fujihara  2   3 , Keizo Tokuhiro  4 , Thomas X Garcia  5   6   7 , Martin M Matzuk  5   6 , Masahito Ikawa  1   2   8
Affiliations

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice

Yuki Oyama et al. Asian J Androl. 2022 May-Jun.

Abstract

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.

Keywords: CRISPR/Cas9; knockout mice; male infertility; spermatozoa; testis.

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Conflict of interest statement

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Figures

Figure 1
Figure 1
Expression patterns of specific genes in various tissues. (a) Digital PCR shows the average transcripts per million (TPM) value per tissue per gene from published mouse RNA-seq datasets. White = 0 TPM, Black ≥30 TPM. Hprt expression is shown as housekeeping control. (b) Expression patterns of Ldhal6b and Smim9 in mouse tissues using RT-PCR. Actb expression is shown as housekeeping control. MW: molecular weight marker. (c) Digital PCR shows the average TPM value per tissue from published human RNA-seq datasets. PLSCR1 and PLSCR2 are orthologs of mouse 1700057G04Rik. C1orf141 is an ortholog of mouse 4921539E11Rik. White = 0 TPM, Black ≥30 TPM. GAPDH expression is shown as housekeeping control. RT-PCR: reverse transcription PCR; Hprt: hypoxanthine guanine phosphoribosyl transferase; Actb: actin beta; C1orf141: chromosome 1 open reading frame 141; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PLSCR1: phospholipid scramblase 1; PLSCR2: phospholipid scramblase 2. The expansions of the other genes have been described in the notes of Table 1
Figure 2
Figure 2
Phenotypic analysis of 4921539E11Rik KO male mice. (a) KO strategy of 4921539E11Rik. Two gRNAs (#2 and #8) were designed to target intron 1 and exon 9. Fw1/2: forward primers for genotyping; Rv1/2: reverse primers for genotyping. (b) Genotyping of 4921539E11Rik KO mice through PCR using primer sets Fw1-Rv1 and Fw2-Rv2, and subsequent Sanger sequencing. MW: molecular weight marker. (c) Comparison of testis size between WT and 4921539E11Rik KO mice. Scale bar = 3 mm. (d) Average testicular weight. Number of males = 3 each. (e) Histological analysis of testes in WT and 4921539E11Rik KO mice. Scale bar = 100 μm. (f) Spermatozoa collected from the cauda epididymis of WT and 4921539E11Rik KO mice. Scale bar = 50 μm. (g) Percentages of motile spermatozoa in WT and 4921539E11Rik KO mice. Sperm motility was measured at 10 min and 120 min after incubation in TYH medium. Number of males = 3 each. KO: knockout; WT: wild type; NS: not significant.
Figure 3
Figure 3
Phenotypic analysis of 4930558C23Rik KO male mice. (a) KO strategy of 4930558C23Rik. Two gRNAs (#3 and #12) were designed to target before and after the protein-coding region. Fw1: forward primer for genotyping; Rv1: reverse primer for genotyping. (b) Genotyping of 4930558C23Rik KO mice through PCR using primer sets Fw1-Rv1 and subsequent Sanger sequencing. MW: molecular weight marker. (c) Comparison of testis size between WT and 4930558C23Rik KO mice. Scale bar = 3 mm. (d) Average testicular weight. Number of males = 3 each. (e) Histological analysis of testes in WT and 4930558C23Rik KO mice. Scale bar = 100 μm. (f) Spermatozoa collected from the cauda epididymis of WT and 4930558C23Rik KO mice. Scale bar = 50 μm. (g) Percentages of motile spermatozoa in WT and 4930558C23Rik KO mice. Sperm motility was measured at 10 min and 120 min after incubation in TYH medium. Number of males = 3 each. KO: knockout; WT: wild type; NS: not significant.
Figure 4
Figure 4
Phenotypic analysis of Tmem210 KO male mice. (a) KO strategy of Tmem210. Two gRNAs (#1 and #2) were designed to target within the 5’-UTR and within exon 4. Fw1: forward primer for genotyping; Rv1/Rv2: reverse primers for genotyping. (b) Genotyping of Tmem210 KO mice through PCR using primer sets Fw1-Rv1 and Fw1-Rv2 and subsequent Sanger sequencing. MW: molecular weight marker. (c) Comparison of testis size between WT and Tmem210 KO mice. Scale bar = 3 mm. (d) Average testicular weight. Number of males = 3 each. (e) Histological analysis of testes in WT and Tmem210 KO mice. Scale bar = 100 μm. (f) Spermatozoa collected from the cauda epididymis of WT and Tmem210 KO mice. Scale bar = 50 μm. (g) Percentages of motile spermatozoa in WT and Tmem210 KO mice. Sperm motility was measured at 10 min and 120 min after incubation in TYH medium. Number of males = 3 each. Tmem210: transmembrane protein 210; KO: knockout; WT: wild type; NS: not significant.
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