{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Heeb LV"],"funding":["Swiss National Science Foundation","European Research Council"],"pagination":["e4724"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10366997"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["13(14)"],"pubmed_abstract":["The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets."],"journal":["Bio-protocol"],"pubmed_title":["HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice."],"pmcid":["PMC10366997"],"funding_grant_id":["407940","756017","101100460","206465"],"pubmed_authors":["Gupta A","Beffinger M","Heeb LV","Taskoparan B","Kobold S","Clavien PA","Katsoulas A","Berg JV"],"additional_accession":[]},"is_claimable":false,"name":"HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice.","description":"The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.","dates":{"release":"2023-01-01T00:00:00Z","publication":"2023 Jul","modification":"2025-04-04T19:47:15.448Z","creation":"2025-04-04T19:47:15.448Z"},"accession":"S-EPMC10366997","cross_references":{"pubmed":["37497456"],"doi":["10.21769/BioProtoc.4724"]}}