Fig 2: Analysis of CTSB gene editing in confirmed REHcas9 cells with CTSB knockout by sequencing. (A) Analysis of gene editing by sequencing of REHcas9 + sgRNA-1 and REHcas9 + sgRNA-4. The plasmids obtained from TOPO + PCR constructs were sequenced using the TOPO M13 vector primer and aligned with the CTSB gene sequence (NG_009217.2) using Geneious® 11.1.5 software. (B) Human CTSB protein sequence (Uniprot code: P07858). The signal peptide sequence is identified in red; the propeptide sequence is identified in blue; the mature protein sequence is identified in black; the C-terminal propeptide sequence is identified in green. The arrows represent the position at the beginning of the changes in the amino-acid sequence caused by the editing of sgRNA-1 (orange arrow) and sgRNA-4 (purple arrow).

Fig 3: In vitro cytotoxicity assay of CTSB KO cells in the presence of ASNase. REHcas9 (uninfected) and REHcas + sgRNA-1 cells were treated with different concentrations of EcA_WT, ErA_WT, and ErA_DM enzymes for 72 h. Cells without treatment and with the addition of the buffer in which the enzymes were diluted are named “control”. The assay was performed in analytical triplicate, and the data represent the mean ± standard deviation (n = 3).

Fig 4: Monitoring of REHcas9 cell infection and CTSB edition. (A) Representative image of REHcas9 infection using lentivirus particles sgRNA-Rosa26. The same procedure was carried out for all lentiviruses produced (sgRNA-1–5 for CTSB gene and sgRNA-RPA3). The infection was confirmed by GFP expression in inverted fluorescence microscope. The figure shows the uninfected cells and infected cells with sgRNA-Rosa26 lentivirus expressing GFP under bright light and green channel excitation at 488 nm. Scale bar, 100 µm. (B) Schematic representation of gene and qPCR-based assay to estimate gene editing efficiency using CRISPR/Cas9 editing tool. Five different primers pairs were designed to flank the target region for the knockout of each sgRNA to validate the editing of the CTSB gene by qPCR. (C) Confirmation of CTSB gene editing by qPCR. Derivate melting curves from qPCR of REHcas9 cells infected with different sgRNAs. The genomic DNA of each cell was used for CTSB editing validation. Uninfected REHcas9 cells and REHcas9 cells infected with sgRNA-Rosa26 (REHcas9 + sgRNA-Rosa26) were used as a negative control (no editing in CTSB gene). The specific primer for the cyclin-dependent kinase 7 (CDKL7) gene was used as an internal control.

Fig 5: Evaluation of CTSB protein amount and activity in REHcas9 + sgRNAs. (A) Western blotting assay image using REHcas9 + sgRNAs. Uninfected REHcas9 and REHcas9 + sgRNAs cells were lysed, 150 µg of total protein was used in each sample, and the film was exposed for 2.5 and 5 min. a-Tubulin protein (60 kDa) was used as a total protein load control. (B) Cathepsin B activity assay of uninfected (in the presence or absence of CTSB inhibitor) and infected cells with sgRNAs (1–5) and Rosa26 using the Cathepsin B Activity Fluorometric Assay Kit (BioVision, Waltham, MA, USA). (C) CTSB activity assay using Sigma-Aldrich reagents (see Section 4) for analysis of lysates of REHcas9 cells and cells infected with sgRNAs-1, 2, and 4 and Rosa26. Results represent the mean ± standard deviation of experiments performed in triplicate. One-way ANOVA statistical analysis followed by Tukey’s post hoc test showed a significant difference (p < 0.05) between the CTSB activities of sgRNA-infected cells (1–5) when compared to the uninfected control REHcas9 cells (*) or when compared to cells treated with CTSB inhibitor (#).