Background

Programmed cell death ligand 1 (PD-L1), also known as CD274, is known to control adaptive immune responses during various pathological conditions such as autoimmune diseases, infections, and cancer (Francisco et al., 2010; Jubel et al., 2020). In particular, higher expression of PD-L1 on antigen-presenting cells as well as on cancer cells is known to engage with PD-1 on activated CD8⁺ T cells, thereby inhibiting their cancer response (Han et al., 2020). Thus, the study of the role of PD-L1 in cancer development and progression was and still is of utmost importance. For this, loss-of-function mouse mutants are invaluable tools whose generation had been time and resource intense for decades.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) is an RNA-guided nuclease system that has been adapted to be a potent gene editing tool. It has quickly evolved to be the method of choice for targeted gene editing and genome-wide screens. It is superior to previously used methodologies such as homologous recombination in embryonic stem cells or use of ZNF (zinc fingers) (Lee et al., 2010; Meyer et al., 2010; Söllü et al., 2010) and TALENs (transcription activator-like effector nucleases) (Cermak et al., 2011; Zhang et al., 2011). Compared to ZNF and TALENs, it relies on DNA–RNA heteroduplex formation rather than protein–DNA interaction (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013). Here, we show a protocol using the CRISPR/Cas9 system to specifically knockout PD-L1 in the C57BL/6 mouse. After the CRISPR/Cas9-mediated dsDNA break, cell-intrinsic DNA repair mechanisms such as non-homologous end joining (NHEJ) and homology-directed repair (HDR) will take place. During NHEJ-based repair, the CRISPR/Cas9-induced double-strand break is repaired by random insertion or deletion of nucleotides at the cut site, which should in theory result in two out of three cases to a frameshift and premature translational stop. This method enables simple and fast generation of loss-of-function alleles and has been rapidly adapted to generate mouse mutants but does not generate precise gene edits. Compared to NHEJ, which leads to random heterogenous outcomes and requires sequencing of the targeted locus when screening founder animals, HDR is template based and more precise, allowing the insertion of specific sequences into the DNA break (Miyaoka et al., 2016; Yang et al., 2020). Here, we show the targeted deletion of exon 3 by insertion of a HindIII restriction site that leads to a defined stop-codon by homologous recombination. This leads to a functional knockout of PD-L1 and can be easily screened by PCR amplification of the targeted locus and a subsequent restriction digest. This protocol can be adapted to target PD-L1 in other mouse strains or cell lines and, most importantly, to target other genes of interest.