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Scientists Create CRISPR/Cas9 Knock-In Mutations in Human T Cells

NEW YORK (GenomeWeb) – Scientists have found a way to add genes to and delete genes from T cells using CRIPSR/Cas9 genome editing, according to a new study published today in the Proceedings of the National Academy of Sciences.

Led by Kathrin Schumann and Alexander Marson from the University of California, San Francisco, and Steven Lin and Jennifer Doudna from the University of California, Berkeley, the scientists were able to generate knock-in mutations in human T cells using Cas9 ribonucleoproteins (RNPs).

The RNPs are preassembled structures consisting of a Cas9 nuclease and the guide RNA (gRNA). Rather than introduce plasmids encoding Cas9 and the gRNAs into the cell using a viral vector, the scientists used electroporation to introduce the RNPs into the cell.

"We tried for a long time to introduce Cas9 with plasmids or lentiviruses, and then to express separately the single-guide RNA in the cell," Schumann, a postdoc in Marson's laboratory, said in a statement. "Using RNPs made outside the cell, so that the cell is responsible for as little of the process as possible, has made a big difference." 

The simplest way to perform CRISPR/Cas9 genome editing is to make a double-stranded break and allow the crude repair mechanism of non-homologous end joining to create a knockout. Alternatively, researchers can also leverage the homology-directed repair (HDR) pathway to create a knock in if a repair template is provided. But getting genome editing to work in human T cells — where there is great potential for immunotherapies for cancer and HIV/AIDS, among other areas — has been "a notable challenge," Marson said, adding he and his colleagues have spent the past year-and-a-half "trying to optimize editing in functional T cells."

The researchers showed that the Cas9 RNPs could not only edit cells' genomes to remove CXCR4 and PD-1 receptors, but also perform the HDR necessary for knock-in editing, reaching knock-in efficiencies of about 20 percent using Cas9 RNPs. HIV uses CXCR4 receptors to infiltrate T cells and PD-1 is an immune checkpoint exploited by cancers to evade apoptosis. The researchers said that the specific pairing of programmed Cas9 RNPs and corresponding HDR templates are required to selectively edit T cell genomes at the nucleotide level. They also added that fluorescence-activated cell sorting (FACS) had the potential to be a useful tool in T cell editing because it could enrich the population of edited cells. 

On the research side, the authors suggested that T cell editing could help scientists with experiments on transcription factors, cis-regulatory elements, and genes implicated in T cell function. Of course, there are potential therapeutic applications for T cell editing and the authors said their findings supported that notion.

"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."