RNA-targeting

RNA-targeting predicts Cas13d (CasRx/RfxCas13d) guide RNA efficiency and evaluates Cas13d orthologs to enable precise RNA perturbation with reduced cellular toxicity in mammalian systems.

Key Features:

• Guide Efficiency Prediction: Predicts CasRx (RfxCas13d) guide RNA efficiency across all human and mouse RefSeq genes based on an analysis of over 127,000 guide RNAs.
• Machine Learning Models: Evaluates three linear models, two ensemble models, and two deep learning models for guide efficiency prediction.
• Deep Learning Insights: The deep learning model identifies specific sequence motifs and secondary features that contribute to high-efficiency guides at spacer positions 15-24.
• Orthogonal Validation: Validates the prediction algorithm across multiple human cell types using orthogonal experiments.
• Custom Sequence Input: Accepts custom target sequences for CasRx guide design.
• Novel Ortholog Discovery: Incorporates metagenomic mining results that identified 46 novel Cas13d orthologs, including DjCas13d with low cellular toxicity and high specificity in human embryonic stem cells, neural progenitor cells, and neurons.
• Generalization Across Orthologs: Generalizes the CasRx-trained guide efficiency model to the DjCas13d ortholog.

Scientific Applications:

• Transcriptome engineering: Enables precise RNA knockdown for transcriptome engineering with reduced off-target effects.
• Functional genomics: Facilitates discovery of gene function through targeted transcript perturbation.
• Disease modeling: Supports modeling diseases by enabling specific RNA perturbations.
• RNA therapeutics development: Assists design of high-efficiency, low-toxicity guide RNAs for therapeutic applications.

Methodology:

Evaluated three linear models, two ensemble models, and two deep learning models on a dataset of over 127,000 guide RNAs; the deep learning model identified sequence motifs and secondary features at spacer positions 15-24; metagenomic mining identified 46 novel Cas13d orthologs; the prediction algorithm was validated via orthogonal experiments across multiple human cell types.