Pgm t

Cd-contaminated soil was collected from a former industrial site in Hunan Province (27°46′N, 112°52′E) and analyzed for Cd content through acid digestion followed by the use of a 7700 × inductively coupled plasma mass spectrometer (Agilent Technologies, Tokyo, Japan). To isolate Cd-resistant and bio-safe bacteria, aerobic Bacillus sp. were isolated from the soil by plating on LB agar plates containing progressively higher concentrations of cadmium chloride (0, 0.5, 1 and 2 mM). Then, in order to isolate aerobic Bacillus sp., the bacterial enrichment cultures were heat-shocked at 80 °C for 20 min. Bacterial genomic DNA was isolated using the TIANamp Bacteria DNA kit (TIANGEN Biotech). The 16S rRNA gene was amplified from the extracted DNA using the universal primers 16S rRNA-F/16S rRNA-R (Additional file 2: Table S2) and the amplification products were cloned in the pGM-T (TIANGEN Biotech) vector using competent E. coli TOP10 cells (TIANGEN Biotech). Sequencing was carried out using T7 and SP6 primers and compared to the GenBank database using the NCBI BLAST program.

Potential off-target sites (POTS) of the sgRNAs were predicted using the online CRISPR design tool (http://crispr.mit.edu/). The top five POTS that were selected. The primers (supplementary Table S2) were used to test whether the sgRNAs have off target mutations and the products were analyzed by both T7E1 assay and sanger sequence cloned into the pGM-T (Tiangen, Beijing, China).

The POTS of the two sgRNAs were predicted using the CRISPR design tool (http://tools.genomeengineering.org). The top 5 POTS were selected for each sgRNA according to ranking scores. The PCR primers used in this study are shown in Supplementary Table S2. The PCR products were analyzed by T7EI assay and then cloned into the pGM-T (Tiangen, Beijing, China) for Sanger sequence.

The PCR amplification product of the 16S rRNA gene (BCC, GenBank accession number: LC496395) was cloned into the vector pGM-T (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The primers used for amplification are shown in Table 1. The concentration of plasmid was detected using a NanoDrop spectrophotometer. Then, the standard recombinant plasmids were prepared at 10-fold dilutions ranging from 10⁶ copies/µL to 10⁰ copies/µL for sensitivity analysis. The plasmid concentration and copy number were determined using the formula: DNA copy number (copy number/µL) = [6.02×10²³ × plasmid concentration (ng/µL) ×10⁻⁹]/[DNA length (in nucleotides) × 660]. The length of the DNA (3226 bp) was the sum of the plasmid fragment length (211 bp) and the vector length (3015 bp).

Total RNA from peripheral blood mononuclear cells (PBMCs) were extracted and reversely transcribed from the carrier (II-1) and a healthy control. The RT-PCR were performed with the primers SH2D1A-RT-F: 5’-CCAGGCGTGTACTGCCTATG-3’ and SH2D1A-RT-R: AGCTGAGGACTTCTTCTCAACTG. The amplified DNA fragments were directly subcloned into the TA cloning vector pGM-T (TIANGEN Biotech, Beijing, China) and sequenced by using the universal sequencing T7 primer.