Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating technology that can be programmed to induce double-strand break (DSB) in the genome wherever needed. After nuclease cleavage, DSBs can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway. For producing targeted gene knock-in or other specific mutations, DSBs should be repaired by the HDR pathway. While NHEJ can cause various length insertions/deletion mutations (indels), which can lead the targeted gene to lose its function by shifting the open reading frame (ORF). Furthermore, HDR has low efficiency compared with the NHEJ pathway. In order to modify the gene precisely, numerous methods arose by inhibiting NHEJ or enhancing HDR, such as chemical modulation, synchronized expression, and overlapping homology arm. Here we focus on the efficiency and other considerations of these methodologies.

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Chronic obstructive pulmonary disease (COPD) in the U.S. is primarily caused by cigarette smoking. COPD patients are highly susceptible to respiratory infections in part due to alveolar macrophage dysfunction despite a substantial increase in macrophages in the lung. Cadmium (Cd) is a toxic metal that is concentrated within tobacco and accumulates in the lung of smokers. We hypothesized that Cd uptake into macrophages alters immune function thereby impairing the macrophage response to invading pathogens. Our hypothesis was tested by comparing primary human monocytes and macrophages, primary mouse bronchoalveolar lavage myeloid cells, and related cell lines. Strikingly, Cd exposure followed by LPS stimulation resulted in a dose-dependent, significant decrease in nuclear p65 activity in macrophages that was not observed in monocytes. This corresponded with Cd-mediated inhibition of IKKβ and an impaired ability to transcribe and release cytokines in response to LPS challenge in vivo. These findings provide novel evidence that Cd has the capacity to disrupt macrophage immune function compared with monocytes. Importantly, Cd results in immune dysfunction in macrophages through inhibition of the NF-κB signaling pathway. Based on these findings, we provide new evidence that Cd contributes to immune dysfunction in the lung of COPD subjects and may increase susceptibility to infection.

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