Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning.

Transcriptome engineering applications in living cells with RNA-targeting CRISPR effectors depend on accurate prediction of on-target activity and off-target avoidance. Here we design and test ~200,000 RfxCas13d guide RNAs targeting essential genes in human cells with systematically designed mismatches and insertions and deletions (indels). We find that mismatches and indels have a position- and context-dependent impact on Cas13d activity, and mismatches that result in G-U wobble pairings are better tolerated than other single-base mismatches. Using this large-scale dataset, we train a convolutional neural network that we term targeted inhibition of gene expression via gRNA design (TIGER) to predict efficacy from guide sequence and context. TIGER outperforms the existing models at predicting on-target and off-target activity on our dataset and published datasets. We show that TIGER scoring combined with specific mismatches yields the first general framework to modulate transcript expression, enabling the use of RNA-targeting CRISPRs to precisely control gene dosage.

Single cell analysis reveals immune cell-adipocyte crosstalk regulating the transcription of thermogenic adipocytes.

Immune cells are vital constituents of the adipose microenvironment that influence both local and systemic lipid metabolism. Mice lacking IL10 have enhanced thermogenesis, but the roles of specific cell types in the metabolic response to IL10 remain to be defined. We demonstrate here that selective loss of IL10 receptor α in adipocytes recapitulates the beneficial effects of global IL10 deletion, and that local crosstalk between IL10-producing immune cells and adipocytes is a determinant of thermogenesis and systemic energy balance. Single Nuclei Adipocyte RNA-sequencing (SNAP-seq) of subcutaneous adipose tissue defined a metabolically-active mature adipocyte subtype characterized by robust expression of genes involved in thermogenesis whose transcriptome was selectively responsive to IL10Rα deletion. Furthermore, single-cell transcriptomic analysis of adipose stromal populations identified lymphocytes as a key source of IL10 production in response to thermogenic stimuli. These findings implicate adaptive immune cell-adipocyte communication in the maintenance of adipose subtype identity and function.