Deep learning and CRISPR-Cas13d ortholog discovery for optimized RNA targeting.

Effective and precise mammalian transcriptome engineering technologies are needed to accelerate biological discovery and RNA therapeutics. Despite the promise of programmable CRISPR-Cas13 ribonucleases, their utility has been hampered by an incomplete understanding of guide RNA design rules and cellular toxicity resulting from off-target or collateral RNA cleavage. Here, we quantified the performance of over 127,000 RfxCas13d (CasRx) guide RNAs and systematically evaluated seven machine learning models to build a guide efficiency prediction algorithm orthogonally validated across multiple human cell types. Deep learning model interpretation revealed preferred sequence motifs and secondary features for highly efficient guides. We next identified and screened 46 novel Cas13d orthologs, finding that DjCas13d achieves low cellular toxicity and high specificity-even when targeting abundant transcripts in sensitive cell types, including stem cells and neurons. Our Cas13d guide efficiency model was successfully generalized to DjCas13d, illustrating the power of combining machine learning with ortholog discovery to advance RNA targeting in human cells.

Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome.

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

Rapid detection of SARS-CoV-2 RNA in saliva via Cas13.

Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies µl-1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.

Rapid, point-of-care molecular diagnostics with Cas13.

Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/µL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.

Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism Measurements.

A protein's folding or function depends on its mobility through the viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity-the viscosity on a time and distance scale that is important for protein folding/function movements. A quasi-null, ultrasensitive time-resolved linear dichroism (TRLD) spectroscopy is proving to be a useful tool for measurements of viscosity on this scale, with previous in vitro studies reporting on the microviscosities of crowded environments mimicked by high concentrations of different macromolecules. This study reports the microviscosity experienced by myoglobin in the E. coli cell's heterogeneous cytoplasm by using TRLD to measure rotational diffusion times. The results show that photolyzed deoxyMb ensembles randomize through environment-dependent rotational diffusion with a lifetime of 34 ± 6 ns. This value corresponds to a microviscosity of 2.82 ± 0.42 cP, which is consistent with previous reports of cytoplasmic viscosity in E. coli. The results of these TRLD studies in E. coli (1) provide a measurement of myoglobin mobility in the cytoplasm, (2) taken together with in vitro TRLD studies yield new insights into the nature of the cytoplasmic environment in cells, and (3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity.