HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice

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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 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.

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