CRISPR‐Cas enzymes: The toolkit revolutionizing diagnostics

Verosloff, Matthew S.; Shapiro, Sarah J; Hawkins, Elizabeth; Alpay, Emel; Verma, Deepika; Stanfield, Emma; Kreindler, Lior; Jain, Sonal; McKay, Bridget; Hubbell, Shae A; Hendriks, Carley G; Blizard, Benjamin A; Broughton, James P.; Chen, Janice S.

doi:10.1002/biot.202100304

Cited by 6 publications

(7 citation statements)

References 43 publications

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“…Virus whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) genotyping are commonly used to identify variants ( 16 , 18 ), but can be limited by long turnaround times and/or the requirement for bulky and expensive laboratory instrumentation. Diagnostic assays based on clustered interspaced short palindromic repeats (CRISPR) ( 19 ) have been developed for rapid detection of SARS-CoV-2 in clinical samples ( 13 , 20 – 23 ), and a few have obtained emergency use authorization (EUA) by the U.S. Food and Drug Administration (FDA) ( 24 – 26 ). Some advantages of these assays for use in laboratory and point-of-care settings include low cost, minimal instrumentation, and a sample-to-answer turnaround time of under 2 h ( 20 , 23 , 27 – 29 ).…”

Section: Introductionmentioning

confidence: 99%

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

et al. 2022

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Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene.

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Section: Introductionmentioning

confidence: 99%

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

et al. 2022

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“…Most recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) technology is revolutionizing the field of nucleic acid detection and next-generation molecular diagnostics, making possible field-deployable POC testing solutions (Myhrvold et al, 2018; Verosloff et al, 2021). CRISPR-Cas systems, naturally occurring in many bacteria and archaea as adaptive immune systems (Wiedenheft et al, 2012), are aiding in the development of novel nucleic acid detection platforms for major diseases such as cancer (Chen et al, 2018) and infectious diseases (Gootenberg et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Novel CRISPR-based detection of Leishmania species

Dueñas

Nakamoto

Cabrera-Sosa

et al. 2022

Preprint

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Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods lead to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. CRISPR-Cas systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with L. (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

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“…Virus whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) genotyping are commonly used to identify variants 16,18 , but can be limited by long turnaround times and/or the requirement for bulky and expensive laboratory instrumentation. Diagnostic assays based on clustered interspaced short palindromic repeats (CRISPR) 19 have been developed for rapid detection of SARS-CoV-2 in clinical samples 13,[20][21][22][23] , and a few have obtained Emergency Use Authorization (EUA) by the US Food and Drug Administration (FDA) [24][25][26] . Some advantages of these assays for use in laboratory and point of care settings include low cost, minimal instrumentation, and a sample-to-answer turnaround time of under 2 hours 20,23,[27][28][29] .…”

Section: Introductionmentioning

confidence: 99%

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

Fasching

Servellita

McKay

et al. 2021

Preprint

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Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants have the potential to guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here we present the development and validation of a COVID-19 variant DETECTR® assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K, and N501Y mutations associated with nearly all circulating viral lineages. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all three targeted SNP mutations. We developed a data analysis pipeline for CRISPR-based SNP identification using the assay from 91 clinical samples (Ct < 30), yielding an overall SNP concordance and agreement with SARS-CoV-2 lineage classification of 100% compared to viral whole-genome sequencing. These findings highlight the potential utility of CRISPR-based mutation detection for clinical and public health diagnostics.

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CRISPR‐Cas enzymes: The toolkit revolutionizing diagnostics

Cited by 6 publications

References 43 publications

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

Novel CRISPR-based detection of Leishmania species

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

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