CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics.

Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.

Distribution and phasing of sequence motifs that facilitate CRISPR adaptation.

CRISPR-associated proteins (Cas1 and Cas2) integrate foreign DNA at the "leader" end of CRISPR loci. Several CRISPR leader sequences are reported to contain a binding site for a DNA-bending protein called integration host factor (IHF). IHF-induced DNA bending kinks the leader of type I-E CRISPRs, recruiting an upstream sequence motif that helps dock Cas1-2 onto the first repeat of the CRISPR locus. To determine the prevalence of IHF-directed CRISPR adaptation, we analyzed 15,274 bacterial and archaeal CRISPR leaders. These experiments reveal multiple IHF binding sites and diverse upstream sequence motifs in a subset of the I-C, I-E, I-F, and II-C CRISPR leaders. We identify subtype-specific motifs and show that the phase of these motifs is critical for CRISPR adaptation. Collectively, this work clarifies the prevalence and mechanism(s) of IHF-dependent CRISPR adaptation and suggests that leader sequences and adaptation proteins may coevolve under the selective pressures of foreign genetic elements like plasmids or phages.

Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.

TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy.

Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr-double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.