In a project spearheaded by investigators at UC San Francisco, scientists have devised a new strategy to precisely modify human T cells using the genome-editing system known as CRISPR/Cas9. Because these immune-system cells play important roles in a wide range of diseases, from diabetes to AIDS to cancer, the achievement provides a versatile new tool for research on T cell function, as well as a path toward CRISPR/Cas9-based therapies for many serious health problems.
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.
The CRISPR/Cas9 system has captured the imagination of both scientists and the general public, because it makes it possible to easily and inexpensively edit genetic information in virtually any organism. T cells, which circulate in the blood, are an obvious candidate for medical applications of the technology, as these cells not only stand at the center of many disease processes, but could be easily gathered from patients, edited with CRISPR/Cas9, then returned to the body to exert therapeutic effects.
Significance
T-cell genome engineering holds great promise for cancer immunotherapies and cell-based therapies for HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been inefficient. We achieved efficient genome editing by delivering Cas9 protein pre-assembled with guide RNAs. These active Cas9 ribonucleoproteins (RNPs) enabled successful Cas9-mediated homology-directed repair in primary human T cells. Cas9 RNPs provide a programmable tool to replace specific nucleotide sequences in the genome of mature immune cells—a longstanding goal in the field. These studies establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.
Efficient editing ofCXCR4in primary human CD4+T cells. (A) Experimental scheme of Cas9:single-guide RNA ribonucleoprotein (Cas9 RNP) delivery toprimary human CD4+T cells for genome editing, followed by genetic and phenotypic characterization. (B) Schematic representation of sgRNA target (blue)and PAM (green) sequence designed to edit coding sequence in the humanCXCR4locus. (C) FACS plots show increasing percentages of cells with low CXCR4expression (CXCR4lo) with higher concentrations of CXCR4 Cas9 RNP (Cas9 RNPlo:0.9μM; Cas9 RNPhi:1.8μM) compared with control-treated cells (Cas9without sgRNA, CTRL; final concentration: 1.8μM). (D) T7 endonuclease I (T7E1) assay demonstrates genome editing in theCXCR4locus with more editingobserved in FACS-sorted CXCR4locells than in CXCR4hicells. Expected PCR product size (938 nt) and approximate expected sizes of T7E1-digested fragmentsare indicated. The total editing frequencies are indicated as percentage of Total Edit below the agarose gel image. (E) Mutation patterns detected by cloningand Sanger sequencing of theCXCR4locus in sorted Cas9 RNP (1.8μM)-treated CXCR4hiand CXCR4locells are compared with the sequence from CXCR4locontrol-treated cells (CTRL). Reference (REF) sequence is shown on top of clonal sequences from each population with sgRNA target (blue) and PAM (green)sequences indicated. Red dashes denote deleted bases, and red sequences indicate mutated nucleotides. Arrowhead indicates the predicted Cas9 cut site. Poor quality sequences obtained from three additional CXCR4 clones were removed from the sequence alignment
PNAS - Generation of knock-in primary human T cells using Cas9 ribonucleoproteins
Full paper with supporting information is here
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