An instrument-free malaria diagnostic

ABSTRACT We propose to develop a sensitive, rapid, and affordable CRISPR-based diagnostic test for malaria parasites. The assay will be simple to run, require no instrumentation or advanced training, and be suitable for resource-limited regions. In the Phase I, we will design and optimize our multi- chambered FAST-CRISPR diagnostic using cultured parasites and plasmid DNA targets. Later in the Phase I or in follow-up studies, we will provide diagnostic kits to laboratories in endemic regions for beta testing. In Specific Aim 1, we will optimize the selection of oligos used in isothermal amplification of targeted regions and optimize the lysis process to expose the DNA. Because reactants from one step of the process flow into the next chamber, we will ensure that buffer compositions are compatible. In Specific Aim 2, we will optimize the CRISPR/Cas12a reactions for activation of the endonuclease activity upon sensing the target sequences. During this Aim, we will assess the use of multiple targets and determine assay specificity and sensitivity. In Specific Aim 3, we will develop the configuration of the FAST device and plan future manufacturing scale-up.