Clustered regularly interspaced short palindromic repeats (CRISPR)-Diagnostics for differential detection of haemoglobin variant S, C, D and E

Although haemoglobin variants are prevalent in low- and middle-income countries, the exact disease burden remains unknown due to a lack of diagnostic capacity. Traditionally, routine clinical haemoglobin variant diagnostics have relied on electrophoresis, which separates the haemoglobins based on size and charge differences. However, electrophoresis-based assays are limited in depth and coverage due to their inability to separate co-migrating variants at the pH employed. Importantly, for genetic counselling of pre-marital couples and prenatal screening of inherited haemoglobinopathy risk, molecular-based assays are required. Here, we leveraged the specificity and dual mismatch intolerance of en31FnCas9 to achieve differential identification of haemoglobin variants S, C, D, and E. Moreover, we demonstrate reliable differential detection of homozygous, heterozygous and compound states of haemoglobin variants due to en31FnCas9s intolerance to dual mismatch in the respective gRNA-target DNA complementarity. Furthermore, we coupled our en31FnCas9-based haemoglobin variant detection to the signal enhancement of recombinase polymerase amplification (RPA) to achieve reliable differential detection of haemoglobin variants from non-invasive saliva and urine samples in <60 minutes. Taken together, our study demonstrates the feasibility of functionalization of CRISPR-based diagnostics as a point-of-care technique towards achieving the democratisation of haemoglobin variant diagnosis in resource-limited settings.