Improved prediction of bacterial CRISPRi guide efficiency from depletion screens through mixed-effect machine learning and data integration.

CRISPR interference (CRISPRi) is the leading technique to silence gene expression in bacteria; however, design rules remain poorly defined. We develop a best-in-class prediction algorithm for guide silencing efficiency by systematically investigating factors influencing guide depletion in genome-wide essentiality screens, with the surprising discovery that gene-specific features substantially impact prediction. We develop a mixed-effect random forest regression model that provides better estimates of guide efficiency. We further apply methods from explainable AI to extract interpretable design rules from the model. This study provides a blueprint for predictive models for CRISPR technologies where only indirect measurements of guide activity are available.

Systematic evaluation of cell-type deconvolution pipelines for sequencing-based bulk DNA methylomes.

DNA methylation analysis by sequencing is becoming increasingly popular, yielding methylomes at single-base pair and single-molecule resolution. It has tremendous potential for cell-type heterogeneity analysis using intrinsic read-level information. Although diverse deconvolution methods were developed to infer cell-type composition based on bulk sequencing-based methylomes, systematic evaluation has not been performed yet. Here, we thoroughly benchmark six previously published methods: Bayesian epiallele detection, DXM, PRISM, csmFinder+coMethy, ClubCpG and MethylPurify, together with two array-based methods, MeDeCom and Houseman, as a comparison group. Sequencing-based deconvolution methods consist of two main steps, informative region selection and cell-type composition estimation, thus each was individually assessed. With this elaborate evaluation, we aimed to establish which method achieves the highest performance in different scenarios of synthetic bulk samples. We found that cell-type deconvolution performance is influenced by different factors depending on the number of cell types within the mixture. Finally, we propose a best-practice deconvolution strategy for sequencing data and point out limitations that need to be handled. Array-based methods-both reference-based and reference-free-generally outperformed sequencing-based methods, despite the absence of read-level information. This implies that the current sequencing-based methods still struggle with correctly identifying cell-type-specific signals and eliminating confounding methylation patterns, which needs to be handled in future studies.