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

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.

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer.

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.

Expression analysis data of BCL11A and γ-globin genes in KU812 and KG-1 cell lines after CRISPR/Cas9-mediated BCL11A enhancer deletion.

The data presented in this article are related to the research article entitled as "Targeted deletion of the BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta-thalassemia disease " [1]. BCL11A is a master regulator of γ-globin gene silencing, and suppresses fetal hemoglobin expression by association with other γ-globin suppressors, and also interacts with human beta-globin locus control region as well as intergenic region between the Aγ and δ-globin genes to reconfigure beta-globin cluster. Thus, HbF reactivation has been proposed to be an approach for the treatment of ß-thalassemia through knockout of BCL11A. Accordingly, an erythroid enhancer sequence was identified that, when inactivated, led to repression of BCL11A and induction of γ-globin in the erythroid lineage [2-7]. This article describes data that obtained from BCL11A gene enhancer modification in KU812 and KG-1 cell lines using the CRISPR-Cas9 genome editing system in order to reactivate γ-globin gene expression.

Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease.

Hemoglobinopathies, such as ß-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of ß-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous ß-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of ß-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating ß-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.