Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR–Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR–Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR–Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR–Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR–Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR–Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

The E antigen of hepatitis B (HBeAg) is positively associated with an increased risk of developing liver cancer and cirrhosis in chronic hepatitis B (CHB) patients. Clinical monitoring of HBeAg provides guidance to the treatment of CHB and the assessment of disease progression. We describe here an affinity binding assay for HBeAg, which takes advantage of G-quadruplex aptamers for enhanced binding and stability. We demonstrate a strategy to improve the binding affinity of aptamers by modifying their sequences upon their G-quadruplex and secondary structures. On the basis of predicting a stable G-quadruplex and a secondary structure, we truncated 19 nucleotides (nt) from the primer regions of an 80-nt aptamer; and the resulting 61-nt aptamer enhanced binding affinity by 19 times (Kd = 1.2 nM). We mutated a second aptamer (40 nt) in one loop region and incorporated pyrrolo-deoxycytidine to replace deoxycytidine in another loop. The modified 40-nt aptamer, with a stable G-quadruplex and two modified loops, exhibited 100 times higher binding affinity for HBeAg (Kd = 0.4 nM) than the unmodified original aptamer. Using the two newly modified aptamers, one serving as the capture and the other as the reporter, we have developed an improved sandwich binding assay for HBeAg. Analyses of HBeAg in serum samples (concentration ranging from 0.1 to 60 ng/mL) of ten hepatitis B patients, showing consistent results with clinical tests, demonstrate a successful application of the aptamer modification strategy and the associated aptamer binding assay.

A 28-year-old woman with a history of asthma and recent deep venous thrombosis presented with fever, chest pain, and peripheral eosinophilia. The patient was subsequently diagnosed with Churg-Strauss syndrome and HIV infection, representing to our knowledge only the second case of this association. Rheumatologic manifestations of HIV may precede clinical signs of infection. This is significant because steroidal and cytotoxic therapy may potentially worsen HIV infection. As the prevalence of HIV infection rises, there may be atypical presentations of various rheumatologic syndromes. The following case demonstrates a patient whose initial presentation for HIV infection was Churg-Strauss syndrome.

Single-cell RNA sequencing (scRNA-seq) provides rich transcriptomic information for studying molecular events and cell heterogeneity at the single-cell level. However, it is challenging to obtain sequence information from rare or low-abundance genes in the presence of other highly abundant genes. We report here a CRISPR-Cas9 technique for the depletion of high-abundance transcripts, resulting in preferential enrichment of rare transcripts. We demonstrate an application of this CRISPR-mediated enrichment technique to scRNA-seq of liver cells infected with hepatitis B virus (HBV). Direct sequencing without the CRISPR-mediated enrichment detected HBV RNA in only 0.6% of the cells. The CRISPR-mediated depletion of the three most abundant transcripts resulted in selective enrichment of the HBV transcript and successful sequencing of HBV RNA in more than 74% of the cells. The improvement enabled a study of HBV infection and interferon treatment of a liver cell model. Gene clusters between the control and HBV-infected Huh7.5-NTCP cells were similar, suggesting that HBV infection did not significantly alter gene expression of the host cells. The treatment with interferon alpha dramatically changed the gene expression of Huh7.5-NTCP cells. These results from the single cell RNA-seq analysis of 7370 cells are consistent with those of bulk experiments, suggesting that HBV is a “stealth virus”.