I am not an expert in this field, but I am very interested and have researched to find an answer. Could you please review the response below to see if it is correct?

Yes, you can use the original lentiCRISPRv2 vector to generate Cas9-expressed cell lines without removing the U6 promoter and the gRNA scaffold region. The presence of the U6 promoter and gRNA scaffold does not cause significant side effects in this context. The U6 promoter drives the expression of gRNA efficiently in mammalian cells [1][2], and studies do not indicate adverse effects from retaining this sequence when the sole purpose is Cas9 expression. Additionally, the U6 promoter's role and efficiency in transcription are well-documented, ensuring stable and predictable outcomes [3][4]. Thus, removal is unnecessary.

Reference

[1] Romanienko, P. J., Giacalone, J., Ingenito, J., Wang, Y., Isaka, M., Johnson, T., You, Y., & Mark, W. H. (2016). A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs. PLoS ONE, 11.

[2] Fry, L., Peddle, C. F., Stevanovic, M., Barnard, A., McClements, M., & MacLaren, R. (2020). Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency.. The CRISPR journal, 3 4, 276-283 .

[3] Chong, S., Hu, P., & Hernandez, N. (2001). Reconstitution of Transcription from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant Polypeptides and a Partially Purified RNA Polymerase III Complex*. The Journal of Biological Chemistry, 276, 20727 - 20734.

[4] Lobo, S., Ifill, S., & Hernandez, N. (1990). cis-acting elements required for RNA polymerase II and III transcription in the human U2 and U6 snRNA promoters.. Nucleic acids research, 18 10, 2891-9 .

I received some assistance from tlooto.com for this response.

I should have read about U6 promoter before having this question. U6 promoter is not likely to be used for long transcripts as I am considering. I think I may continue using empty lentiCRISPRv2 for Cas9 expression. Just don't know why my cells get stressed after transduction and nearly stop growing, perhaps because it is a primary cell line.