Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

The synthetic histone-binding regulator protein PcTF activates interferon genes in breast cancer cells.

BACKGROUND: Mounting evidence from genome-wide studies of cancer shows that chromatin-mediated epigenetic silencing at large cohorts of genes is strongly linked to a poor prognosis. This mechanism is thought to prevent cell differentiation and enable evasion of the immune system. Drugging the cancer epigenome with small molecule inhibitors to release silenced genes from the repressed state has emerged as a powerful approach for cancer research and drug development. Targets of these inhibitors include chromatin-modifying enzymes that can acquire drug-resistant mutations. In order to directly target a generally conserved feature, elevated trimethyl-lysine 27 on histone H3 (H3K27me3), we developed the Polycomb-based Transcription Factor (PcTF), a fusion activator that targets methyl-histone marks via its N-terminal H3K27me3-binding motif, and co-regulates sets of silenced genes. RESULTS: Here, we report transcriptome profiling analyses of PcTF-treated breast cancer model cell lines. We identified a set of 19 PcTF-upregulated genes, or PUGs, that were consistent across three distinct breast cancer cell lines. These genes are associated with the interferon response pathway. CONCLUSIONS: Our results demonstrate for the first time a chromatin-mediated interferon-related transcriptional response driven by an engineered fusion protein that physically links repressive histone marks with active transcription.

Regulation of cancer epigenomes with a histone-binding synthetic transcription factor.

Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously, we reported the first synthetic transcriptional activator, the "Polycomb-based transcription factor" (PcTF) that reads histone modifications through a protein-protein interaction between the polycomb chromodomain motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of the polycomb-based transcription factor fusion protein. Transcriptome and chromatin profiling revealed several polycomb-based transcription factor-sensitive promoter regions marked by distal H3K27me3 and proximal fusion protein binding. These results illuminate a mechanism in which polycomb-based transcription factor interactions bridge epigenomic marks with the transcription initiation complex at target genes. In three cancer-derived human cell lines tested here, some target genes encode developmental regulators and tumor suppressors. Thus, the polycomb-based transcription factor represents a powerful new fusion protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation.

Erratum: Regulation of cancer epigenomes with a histonebinding synthetic transcription factor.

[This corrects the article DOI: 10.1038/s41525-016-0002-3.].