Identification of functional regulatory elements in the human genome using pooled CRISPR screens.

BACKGROUND: Genome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise < 2% of the sequence space in the human genome leaving a substantial portion of the genome uninterrogated. Noncoding regions of the genome harbor important regulatory elements (e.g. promoters, enhancers, silencers) that influence cellular processes but high-throughput methods for evaluating their essentiality have yet to be established. RESULTS: Here, we describe a CRISPR-based screening approach that facilitates the functional profiling of thousands of noncoding regulatory elements in parallel. We selected the tumor suppressor p53 as a model system and designed a pooled CRISPR library targeting thousands of p53 binding sites throughout the genome. Following transduction into dCas9-KRAB-expressing cells we identified several regulatory elements that influence cell proliferation. Moreover, we uncovered multiple elements that are required for the p53-mediated DNA damage response. Surprisingly, many of these elements are located deep within intergenic regions of the genome that have no prior functional annotations. CONCLUSIONS: This work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.

PD-1 Mediated Regulation of Unique Activated CD8 + T Cells by NK Cells in the Submandibular Gland.

The increasing utilization of anti-PD-1 immune checkpoint blockade (ICB) has led to the emergence of immune-related adverse events (irAEs), including sicca syndrome. Interestingly, we found that the submandibular gland (SMG) of PD-1 deficient mice harbors a large population of CD8 + T cells, reminiscing ICB induced sicca. This phenotype was also observed in the SMG of both NK cell-depleted C57BL/6 animals and NK cell-deficient animals. Mechanistically, using mice conditionally deficient for PD-L1 in the NK cell lineage, we discovered that NK cells regulate CD8 + T cell homeostasis via the PD-1/PD-L1 axis in this organ. Importantly, single-cell RNA sequencing of PD-1 deficient SMG CD8 + T cells reveals a unique transcriptional profile consistent with TCR activation. These cells have limited TCR diversity and phenotypically overlap with GzmK + CD8 + T autoimmune cells identified in primary Sjögren's syndrome patients. These insights into NK cell immunoregulation in the SMG, and the consequences of disrupted CD8 + T cell homeostasis, provide opportunities for preventing the development of irAEs. Highlights: Elevated CD8 + T cells in the submandibular gland (SMG) of PD-1 deficient mice parallel sicca-like irAEs seen in ICB patients. In addition to their previously described hyporesponsive phenotype, NK cells in the SMG regulate CD8 + T cell homeostasis through the PD-L1/PD-1 axis. PD-1 deficient SMG CD8 + T cells display unique transcriptional profiles associated with proinflammatory functions, TCR activation, interferon stimulation, and exhaustion. Oligoclonal expansion and similarities in TCR sequences indicate T cell activation and a preference for recognizing specific antigens.

The Yin and Yang of Targeting KLRG1+ Tregs and Effector Cells.

The literature surrounding KLRG1 has primarily focused on NK and CD8+ T cells. However, there is evidence that the most suppressive Tregs express KLRG1. Until now, the role of KLRG1 on Tregs has been mostly overlooked and remains to be elucidated. Here we review the current literature on KLRG1 with an emphasis on the KLRG1+ Treg subset role during cancer development and autoimmunity. KLRG1 has been recently proposed as a new checkpoint inhibitor target, but these studies focused on the effects of KLRG1 blockade on effector cells. We propose that when designing anti-tumor therapies targeting KLRG1, the effects on both effector cells and Tregs will have to be considered.

Optimization of AsCas12a for combinatorial genetic screens in human cells.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.