evidence: According to the results of the T7EI assay, sgRNAs with relatively high efficiency were selected and nine dual-sgRNA combinations, which included those targeting the same protein-coding gene and those targeting different protein-coding genes, were constructed in the lentiviral vector. The sgRNA target sites in these J-Lat 10.6 cells stably expressing Cas9 dual-guide RNAs (gRNAs) were sequenced to analyze the types and frequency of mutations. Nucleotide sequences analysis indicated that almost all of the mutations were clustered near the Cas9 cleavage site, at 3–4 nt upstream of the protospacer adjacent motif (PAM) sequence, confirming that NHEJ repair caused the mutations. The combinations with a high proportion of insertions and deletions of the HIV-1 genome were sg-Gag1-J + sg-Gag2-J (Insertions: 44%; Deletions: 32%), sg-Gag2-J + sg-Pol2-J (Insertions: 19%; Deletions: 63%), and sg-Pol2-J + sg-Tat1-J (Insertions: 54%; Deletions: 25%), respectively (Figure 3). The mutated sequences of the sgRNA-targeted regions in the three most efficient dual-sgRNA combinations are displayed in Figure 4. Mutation sequences of other combinations are shown in Supplementary Figures S1, S2. The incidence of nucleotide substitutions (NSs) was low, only being detected in four out of nine combinations, and at less than 10% (Figures 3C,E,H,I).