The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 10⁷ transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 10⁷ TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.

由于存在 SV40 T 抗原编码序列，使用人胚肾 (HEK) 293T 细胞系制造用于体内应用的载体会引起安全问题。我们使用 CRISPR-Cas9 基因组编辑从 HEK293T 细胞中去除 SV40 T 抗原编码序列，方法是用表达 Cas9 的重组质粒和对应于 T 抗原编码区开头和结尾的两个不同的单引导 RNA (sgRNA) 转染它们。使用 PCR 鉴定缺乏 T 抗原编码序列的细胞克隆。亲代 HEK293T 细胞系的全基因组 (WG) 和靶向位点扩增 (TLA) 测序揭示了多个 SV40 T 抗原编码序列取代了 3 号染色体上的细胞序列。推定的 T 抗原无效克隆表明映射到 T 的序列读取丢失抗原编码序列。对从 T 抗原无效克隆制备的细胞提取物进行蛋白质印迹分析，证实不存在 SV40 大和小 T 抗原蛋白。使用T抗原无效克隆产生的慢病毒载体表现出高达1.5×10 ⁷转导单位(TU)/mL的滴度，而从亲本HEK293T细胞系获得的滴度高达4×10 ⁷ TU/mL。还评估了 T 抗原阴性细胞产生高效价腺相关病毒 (AAV) 载体的能力。获得的结果表明，T 抗原序列的缺失不会影响 AAV 载体滴度。