A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping

Abstract

Background: CRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection. Methods: We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance. Findings: The suite of Plasmodium assays achieved analytical sensitivities ranging from 25-188 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities. Interpretation: These novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance.

Plasmodium knowlesi: the game changer for malaria eradication

“…This is a novel diagnostic approach, where CRISPR technology and RPA assay are combined. SHERLOCK is an ultrasensitive CRISPR-based method that allows the detection of infections from asymptomatic carriers [ 127 , 128 ]. The Isothermal detection tools are more promising than the PCR-based methods.…”

Multi-Omics Advancements towards Plasmodium vivax Malaria Diagnosis

“…CRISPR has shown a high success rate in the diagnosis of viral infectious diseases such as Zika, Dengue [ 96 ], and HPV [ 97 ], and is being tested for SARS-CoV2 [ 98 ] diagnosis. Similarly, potential CRISPR-based panels are being explored to diagnose and differentiate symptomatic from asymptomatic patients [ 99 ], differentiate Plasmodium species, and genotype antimalarial drug resistance [ 100 ] in malaria patients.…”

Technologies for Detection of Babesia microti: Advances and Challenges

“…To date, CRISPR-based diagnostics have not been applied to the detection of Babesia. However, Cunningham et al used SHERLOCK CRISPR collateral cleavage-based diagnostics to develop a fast, low-cost deployable assay capable of Plasmodium detection, species differentiation and drug-resistance genotyping [90]. This CRISPR-based SHERLOCK assay uses an isothermal RPA reaction to generate double-stranded DNA amplicons of the target sequence, in vitro transcription of RPA product to produce single-stranded RNA (ssRNA) targets and the collateral cleavage of fluorescent or colorimetric RNA reporter molecules to produce a detection signal.…”