The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

Mad7 is a CRISPR enzyme, similar to Cas9 and Cas12a, which is of great interest to industry and academia due to its permissive licensing agreement, which states that everyone is free to use it but not distribute it commercially. This paper demonstrates that Mad7 genome editing works in CHO cells and provides the CHO engineering community with the computational tools needed to generate Mad7 compatible gRNAs against the CHO genome.

Mad7 is a CRISPR enzyme, similar to Cas9 and Cas12a, which is of great interest to industry and academia due to its permissive licensing agreement, which states that everyone is free to use it but not distribute it commercially. This paper demonstrates that Mad7 genome editing works in CHO cells and provides the CHO engineering community with the computational tools needed to generate Mad7 compatible gRNAs against the CHO genome.

pronounced inhibition of cell proliferation (p = 0.0046). PI3K inhibition suppressed cancer cell growth, migration and colony formation in vitro. Pan-PI3K inhibition, anti-programmed death 1 (PD1) therapy and combination improved the overall survival (OS) of syngeneic mice with PTEN-deleted tumors from 27 days of the control to 48, 37 and 65 days, respectively. In mice with tumors not containing a PI3K pathway alteration, OS was prolonged by the combination, but not single treatments. Pan-PI3K inhibition significantly upregulated CD80, CD86, MHC-I and MHC-II in dendritic cells, and downregulated the transforming growth factor beta pathway with a false discovery rate (FDR)-adjusted q-value of 0.001. Interferon alpha response was significantly upregulated with anti-PD1 therapy (q value: < 0.001) and combination (q value: 0.027). Compared to the control, combination treatment increased CD8 + T cell infiltration (p = 0.005), decreased T reg cell infiltration (p = 0.036), and upregulated the expression of multiple immunostimulatory cytokines and Granzyme B (p< 0.01). Secondary resistance was associated with upregulation of the mammalian target of rapamycin (mTOR) pathway and multiple Sprr family genes. Conclusions The combination Pan-PI3K inhibition and ICB has significant anti-tumor effects in aUC with or without activated PI3K pathway and warrant further clinical investigation. This combination creates an immunostimulatory tumor milieu. Secondary resistance is associated with upregulation of the mTOR pathway and Sprr family genes. Base on this study, a Phase II clinical trial has been designed.