A large CRISPR-induced bystander mutation causes immune dysregulation.

A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic bystander mutations that escape detection by routine targeted genotyping assays

Understanding human autoimmunity risk within the IL2RA super enhancer

Human disease risk has been linked to hundreds of variants in our DNA. Understanding of these unbiased genetic associations has long held the promise of revealing insights into disease mechanisms. However, disease-risk variants overwhelmingly reside in non-coding sequences – long stretches of our chromosomes that we still know relatively little about. Here I develop new tools and methodologies to understand genetic risk for disease for a critical autoimmunity locus, IL2RA. I first demonstrate that CRISPR activation can be adapted for high throughput enhancer screens. By tiling CRISPR-activation across the super-enhancer within the IL2RA locus, I systematically map functional IL2RA enhancers in the disease-associated non-coding sequences. Undertaking a genetic perturbation approach, I dissect how distinct IL2RA enhancers regulate immune cell function as well as shape risk of autoimmunity in vivo. Using CRISPR-engineered enhancer deletion mice and human immune cells I identified two novel IL2RA enhancers; a maintenance enhancer that controls IL2RA expression in anti-inflammatory regulatory T cells and a disease-associated IL2RA enhancer that controls the timing of IL2RA induction in pro-inflammatory immune cells. Having discovered enhancers that regulate IL2RA in different contexts I interrogated their effects in an in vivo model of autoimmune disease. Deletion of the conserved stimulation-responsive enhancer that harbors a human variant protective against T1D completely protected non-obese diabetic (NOD) mice from diabetes. This work decodes a critical autoimmunity association, develops a cis-regulatory framework at the IL2RA locus, and causally links IL2RA gene regulation to autoimmunity. The tools and strategies developed in these studies can be used to decode disease-associated loci in the human genome

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells