A CRISPR-based assay for the detection of opportunistic infections post-transplantation and for the monitoring of transplant rejection

Abstract

In organ transplantation, infection and rejection are major causes of graft loss linked by the net state of immunosuppression. To diagnose and treat these conditions earlier, and to improve long-term patient outcomes, refined strategies for the monitoring of patients after graft transplantation are needed. Here, we show that a fast and inexpensive assay based on CRISPR-Cas13 accurately detects BK polyomavirus DNA and cytomegalovirus DNA from patient-derived blood and urine samples, as well as CXCL9 mRNA (a marker of graft rejection) at elevated levels in urine samples from patients experiencing acute renal-transplant rejection. The assay, which we adapted for lateral-flow readout, enables via simple visualization the post-transplantation monitoring of common opportunistic viral infections and of graft rejection, and should facilitate point-of-care posttransplantation monitoring.

Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria

“…There are many Cas enzymes that could have been used. We chose Cas12a [as opposed to the Cas13 family (21,22), which also has nonspecific nuclease activity] so DNA targets could be directly detected instead of RNA, particularly in DBS where RNA may be degraded. The rapid enzymatic kinetics of Cas12a also make this nucleic acid-based technology comparable to the POC format of antigen-based lateral flow immunoassays.…”

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…In most of these platforms, a commercially available universal test strip, the HybriDetect-Universal Lateral Flow Assay Kit, was used. This dipstick was originally designed for qualitative or even quantitative rapid testing of proteins, antibodies or gene amplicons, but has been adapted to function in several LFA based CRISPR sensing methods ( Bai et al, 2019 ; Chang et al, 2019 ; Gootenberg et al, 2018 ; Kaminski et al, 2020 ; Mukama et al, 2020b ; Sullivan et al, 2019 ; Tsou et al, 2019 ). This platform offers a Streptavidine line as well as an antibody line that can capture anti-FITC coated AuNPs ( Fig.…”

Henceforth CRISPR

“…In fact, CRISPR's exquisite specificity makes it outstanding for the detection of nucleic acids in biofluids. Notably, target genes in buccal samples can be detected electrically via inactivated Cas9 immobilized on a graphene field-effect transistor without the need for amplification 2 , and Cas13 has been used to monitor opportunistic viral infections in blood and urine samples following organ transplantation, and to detect transplant rejection in urine samples 3 . And, most recently, fast and inexpensive lateral-flow fluorescence assays containing Cas12a or Cas13a can detect SARS-CoV-2 RNA accurately in nasopharyngeal samples [4][5][6] .…”