Journal: Scientific Reports

Article Title: Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice

doi: 10.1038/s41598-025-22373-9

Figure Lengend Snippet: Efficacies of CRISPR/Cas9 gene editing targeting Osr1 with two sgRNA constructs. ( A ) Scheme of the sgRNA and primers targeted positions at Osr1 . ( B ) An example of the capillary electrophoresis for blastocysts after injection of Cas9 protein along with two sgRNAs, Osr1a and Osr1b . The Osr1 -WT-1 is the wild-type blastocyst. PCR amplification using Primer 2 and Primer 3 yielded a single 287-bp band from the wild-type blastocyst. Those blastocysts were evaluated by whether they have bands equal to the wild-type embryo (287-bp) or not. If it has only one 287-bp band, we count it as a wild-type. If it has one band at 287 bp and the other drops below it, then it is heterozygous. If it only has bands below 287 bp, it indicates having deletions on both alleles. In this batch of blastocysts, numbers 3, 5, 9, 11, 12, 13, 14, 15, 16, and 18 are found no deletion; number 19 is heterozygous; numbers 1, 2, 4, 6, 7, 8, 10, and 17 are biallelic knockouts. ( C ) The efficiencies of gene editing with two sgRNAs ( Osr1 a and Osr1b )

Article Snippet: In brief, the aliquoted sgRNAs and Cas9 protein (SpCas9 2NLS nuclease, Synthego) were premixed (Cas9 protein: 1 μM; sgRNAs: 2μM; 10 μL in total) and incubated at 37 °C for 15 min to form ribonucleoprotein right before microinjection.

Techniques: CRISPR, Construct, Electrophoresis, Injection, Amplification

Journal: Scientific Reports

Article Title: Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice

doi: 10.1038/s41598-025-22373-9

Figure Lengend Snippet: Establishment of Osr1 knockout mouse ES cells derived from the blastocyst embryos developed from Cas9 protein and 3 sgRNAs injected mouse zygotes. ( A ) Electrophoresis result of 10 possible Osr1 knockout ES cell lines. Primer 2 and Primer 5 were used to amplify 929-bp products from wild-type ES cells. If it has only one 929-bp band, we count it as wild-type. If it has one band at 929 bp and the other drops below it, then it is heterozygous. If it only has bands below 929 bp, it indicates deletions on both alleles. Therefore, ES cell (Lane) #1, 5, 7, and 9 are wild-type; #2, 6 are heterozygous; #3, 4, 8, and 10 are biallelic knockout. The full gel image can be found in the supplementary information (Fig. ). ( B ) Alignment of the sequencing results with the Osr1 gene. The numbers 1 to 10 correspond to Lane 1 to Lane 10 in ( A )

Article Snippet: In brief, the aliquoted sgRNAs and Cas9 protein (SpCas9 2NLS nuclease, Synthego) were premixed (Cas9 protein: 1 μM; sgRNAs: 2μM; 10 μL in total) and incubated at 37 °C for 15 min to form ribonucleoprotein right before microinjection.

Techniques: Knock-Out, Derivative Assay, Injection, Electrophoresis, Sequencing

Journal: Scientific Reports

Article Title: Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice

doi: 10.1038/s41598-025-22373-9

Figure Lengend Snippet: In vivo development of the mouse fetus (E13.5). ( A ) The pronuclear stage embryos were microinjected with CRISPR/Cas9 constructs targeting the Osr1 gene. After embryo transfer, the implanted uterine horns are harvested on day 13.5, showing mostly degenerated fetuses. Scale bar: 2 mm. ( B ) Most of the fetuses show various degrees of degeneration. Scale bar: 1 mm

Article Snippet: In brief, the aliquoted sgRNAs and Cas9 protein (SpCas9 2NLS nuclease, Synthego) were premixed (Cas9 protein: 1 μM; sgRNAs: 2μM; 10 μL in total) and incubated at 37 °C for 15 min to form ribonucleoprotein right before microinjection.

Techniques: In Vivo, CRISPR, Construct

Journal: Scientific Reports

Article Title: Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice

doi: 10.1038/s41598-025-22373-9

Figure Lengend Snippet: Histological examination (HE staining) of E12.5 mouse fetuses pronuclear injected with CRISPR/Cas9 constructs targeting Osr1 gene. ( A ) A wild-type control fetus (without construct injection). ( B ) A CRISPR/Cas9 construct-injected fetus shows no observable kidney development (red dotted circle). h: heart; li: liver; st: stomach; ki: kidney. Scale bar: 200 μm

Article Snippet: In brief, the aliquoted sgRNAs and Cas9 protein (SpCas9 2NLS nuclease, Synthego) were premixed (Cas9 protein: 1 μM; sgRNAs: 2μM; 10 μL in total) and incubated at 37 °C for 15 min to form ribonucleoprotein right before microinjection.

Techniques: Staining, Injection, CRISPR, Construct, Control