The top concern for the clinical use
The top concern for the clinical use of CRISPR-Cas9 technology is safety. Off-target alterations must be avoided to ensure genome integrity and proper cellular function. WGS found no off-target effects among the top 49 predicted off-target sites. WGS did find 4,658 indels and 1,891 SNVs in sgRNA1/2-induced CD4+ AM966 receptor (Figures 3D and 3E), but these were absent in wild-type CD4+ cells. This finding is similar to the Schaefer report, which showed the unexpected mutations in CRISPR-Cas9-edited mice. Schaefer concluded that these mutations were caused by CRISPR-Cas9 editing. However, we also found 4,644 indels and 1,925 SNVs in wild-type CD4+ only, and these were absent in sgRNA1/2-induced CD4+ cells. These two numbers are comparable. Therefore, indels and SNVs showed by WGS could not be concluded to be caused by CRISPR-Cas9 editing. Cellular heterogenicity may be a possible explanation.
Although TIDE analysis and WGS showed no apparent off-target effects, we did find unexpected on-target CCR5 mutations (Figures 1E, 1G, 2D, and 4C). These mutations would sometimes be like CCR5Δ32/Δ32 homozygotes, but with unexpected indels or point mutations. The only way to distinguish these mutations from the expected CCR5Δ32/Δ32 mutation is through sequencing.
Materials and Methods
Conflicts of Interest
Acknowledgments We thank Dr. Lawrence Kleiman for English proofreading and suggestions. This work was supported by the National Natural Science Foundation of China (81571991 to M.W.) and Nankai University starting fund (ZB15006101 to M.W.).
Introduction Macrophages were found in different regions of the human cochlea, e.g., stria vascularis, spiral ligament, peripheral and central nerve bundles, Rosenthal's canal, tympanic covering layer and even in the organ of Corti in steady state (Liu et al., 2018), with some resident macrophages found to be positive for markers CD163, IBA1, and CD68 (O'Malley et al., 2016). Immunohistochemistry studies have yielded similar findings in the cochleae of animals (Warchol, 1997; Hirose et al., 2005; Okano et al., 2008; Warchol et al., 2012) although anatomic differences exist between human and animals. The fact that macrophages reside in both rodent and human cochleae suggests that the inner ear contains immune-competent cells that may participate in both innate and adaptive immune responses. These macrophages trap and process antigens reaching the inner ear by invasion of pathogens, insertion of CI electrode, abnormally exposed autoantigens, hence serving as an immunity domain in the cochlea together with immune cells in the endolymphatic sac which has direct communication with the endolymph of the cochlea. Lymphocytes were found in guinea pig endolymphatic sac (ES) by using electron microscopy in 80's (Rask-Andersen and Stahle, 1979). CD4 and CD8 (cluster of differentiation 4 and 8), the transmembrane glycoproteins of T helper and cytotoxic T cells respectively, are co-receptors of the T cell receptor (TCR). CD4 and CD8 assist the TCR in communicating with antigen-presenting cells. The receptor complex binds to distinct regions of the antigen-presenting major histocompatibility complex (MHC) molecules. While CD4 binds to class II MHC molecule (MHCII), CD8 is specific for the class I MHC protein (MHCI) (Gao and Jakobsen, 2000). CD4 positive (CD4+) T helper cells' main role is to send signals to other types of immune cells, including CD8+ killer cells. The latter then destroy the pathogens by releasing toxic granules containing powerful enzymes which kill the pathogen-infected cells. While we need to know the characteristics of the inner ear resident macrophages and their roles in innate immunoreactivity in the human cochlea by analyzing the molecules expressed, we hope to analyze other cells such as lymphocytes in the cochlea. These lymphocytes in animal cochleae are considered to play roles in adaptive immunity (Yang et al., 2015). However, lymphocytes have not been thoroughly studied in the human cochlea.