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takyon writes:

Nature has a comprehensive analysis and history of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), the disruptive technique that is allowing genetic engineering and gene therapy to flourish:

CRISPR methodology is quickly eclipsing zinc finger nucleases and other [genetic] editing tools (see 'The rise of CRISPR'). For some, that means abandoning techniques they had taken years to perfect. "I'm depressed," says Bill Skarnes, a geneticist at the Wellcome Trust Sanger Institute in Hinxton, UK, "but I'm also excited." Skarnes had spent much of his career using a technology introduced in the mid-1980s: inserting DNA into embryonic stem cells and then using those cells to generate genetically modified mice. The technique became a laboratory workhorse, but it was also time-consuming and costly. CRISPR takes a fraction of the time, and Skarnes adopted the technique two years ago.

Researchers have traditionally relied heavily on model organisms such as mice and fruit flies, partly because they were the only species that came with a good tool kit for genetic manipulation. Now CRISPR is making it possible to edit genes in many more organisms. In April, for example, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported using CRISPR to study Candida albicans, a fungus that is particularly deadly in people with weakened immune systems, but had been difficult to genetically manipulate in the lab. Jennifer Doudna, a CRISPR pioneer at the University of California, Berkeley, is keeping a list of CRISPR-altered creatures. So far, she has three dozen entries, including disease-causing parasites called trypanosomes and yeasts used to make biofuels.

Yet the rapid progress has its drawbacks. "People just don't have the time to characterize some of the very basic parameters of the system," says Bo Huang, a biophysicist at the University of California, San Francisco. "There is a mentality that as long as it works, we don't have to understand how or why it works." That means that researchers occasionally run up against glitches. Huang and his lab struggled for two months to adapt CRISPR for use in imaging studies. He suspects that the delay would have been shorter had more been known about how to optimize the design of guide RNAs, a basic but important nuance.

takyon writes:

We have previously covered CRISPR, its rising popularity, its breakthroughs, and creations.

Now, scientists at UC San Francisco and UC Berkeley have used CRISPR/Cas9 to modify human T cells in order to control immune functions:

Using their novel approach, the scientists 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 group 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 tumors.

[In] practice, editing T cell genomes with CRISPR/Cas9 has proved surprisingly difficult, said Alexander Marson, PhD, a UCSF Sandler Fellow, and senior and co-corresponding author of the new study. "Genome editing in human T cells has been a notable challenge for the field," Marson said. "So we spent the past year and a half trying to optimize editing in functional T cells. There are a lot of potential therapeutic applications, and we want to make sure we're driving this as hard as we can."

[...] 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. He hopes that Cas9-based therapies for T cell-related disorders, which include autoimmune diseases as well as immunodeficiencies such as "bubble boy disease," will enter the clinic in the future. "There's actually well-trodden ground putting modified T cells into patients. There are companies out there already doing it and figuring out the safety profile, so there's increasing clinical infrastructure that we could potentially piggyback on as we work out more details of genome editing," Marson said. "I think CRISPR-edited T cells will eventually go into patients, and it would be wrong not to think about the steps we need to take to get there safely and effectively."

The full paper [PDF] is available.

Original Submission