*Purpose: There is an unmet need in transplantation to identify allograft injury non-invasively. More than 50% of patients with kidney transplants lose their grafts within 10 years. If we better monitor patients for rejection and opportunistic infections, which are the main causes of allograft loss, we could improve transplant survival. Using CRISPR/Cas13 technology, we developed a point-of-care assay that is inexpensive, fast and sensitive to detect the biomarker CXCL9 mRNA, BK virus (BKV) and cytomegalovirus (CMV), allowing better monitoring of T-cell mediated rejection (TCMR) and opportunistic infections after transplantation.

*Methods: We adapted and optimized a CRISPR-Cas13 platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) towards detecting BKV, CMV, and CXCL9 mRNA. We tested for BKV and CMV infection on patient samples (n=118) processed with HUDSON protocol, which avoids the need for time-consuming, column-based sample preparations. For the monitoring of BKV infection, we tested in urine and plasma samples of infected patients and controls. For CMV detection, we tested plasma samples of infected patients and controls. For rejection monitoring, we tested CXCL9 mRNA with the SHERLOCK protocol on urine samples of biopsy-proven kidney rejection and compared to non-rejection/stable kidney allografts.

*Results: For both CMV and BK virus detection, we had 100% sensitivity and 100% specificity compared with the gold standard qPCR (Fig. A). Next, we employed a lateral-flow dipstick as easy-to-read visual output for the biomarker targets (Fig. B). On rejection monitoring, we observed higher CXCL9 levels in the rejection group, compared to the stable transplant patients (p<0.0001). We confirmed CXCL9 upregulation in rejection samples with qPCR as well as ELISA. We also tested for the biomarker in prospective samples of patients experiencing TCMR rejection and compared them with creatinine levels (representative case, Fig. C). We successfully detected increasing CXCL9 levels on the rejection period, as confirmed by biopsy.

*Conclusions: The monitoring of CXCL9 in urine using CRISPR/cas13 constitutes a promising technique for the detection of rejection and opportunistic infections non-invasively. Moreover, persistent elevation of CXCL9 after treatment can also be an indicator of resistant rejection, serving as a marker of treatment response. This low-cost diagnostic assay may allow patients to better monitor for rejection and opportunistic infections and improve transplant outcomes.

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