Researchers at the University of California - San Francisco used the genome-editing tool known as CRISPR/Cas9 to precisely modify human T cells. They were able to disable a protein on the T-cell surface called CXCR4, which can be exploited by HIV when the virus infects T cells and causes AIDS.
The team also successfully shut down PD-1, a protein that has attracted intense interest in the burgeoning field of cancer immunotherapy, as scientists have shown that using drugs to block PD-1 coaxes T cells to attack tumours. The CRISPR/Cas9 system makes it possible to easily and inexpensively edit genetic information in virtually any organism. Cas9, an enzyme in the CRISPR system that makes cuts in DNA and allows new genetic sequences to be inserted, has generally been introduced into cells using viruses or circular bits of DNA called plasmids. Then, in a separate step, a genetic construct known as single-guide RNA, which steers Cas9 to the specific spots in DNA where cuts are desired, is also placed into the cells.
Until recently, editing human T cells with CRISPR/Cas9 has been inefficient, with only a relatively small percentage of cells being successfully modified. While scientists have had some success in switching off genes by inserting or deleting random sequences, they have not yet been able to use CRISPR/Cas9 to paste in (or "knock in") specific new sequences to correct mutations in T cells.
A team led by first authors Kathrin Schumann, a postdoctoral fellow in the lab of Alexander Marson, a UCSF Sandler Fellow, and Steven Lin, a postdoctoral fellow in the lab of University of California, Berkeley's Jennifer Doudna, cracked these problems by streamlining the delivery of Cas9 and single-guide RNA to cells.
In lab dishes, the group assembled Cas9 ribonucleoproteins, or RNPs, which combine the Cas9 protein with single-guide RNA. They then used a method known as electroporation, in which cells are briefly exposed to an electrical field that makes their membranes more permeable, to quickly deliver these RNPs to the interior of the cells.
With these innovations, the researchers successfully edited CXCR4 and PD-1, even knocking in new sequences to replace specific genetic "letters" in these proteins. The group was then able to sort the cells using markers expressed on the cell surface, to help pull out successfully edited cells for research, and eventually for therapeutic use.
Marson stressed that, while recent reports of CRISPR/Cas9 editing of human embryos have stirred up controversy, T cells are created anew in each individual, so modifications would not be passed on to future generations. The study was published in Proceedings of the National Academy of Sciences.
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