CRISPR-Cas13 in malaria parasite: Diagnosis and prospective gene function identification

Quansah, Elvis; Chen, Yihuan; Yang, Shijie; Wang, Junyan; Sun, Dezhi; Zhao, Yujia; Chen, Ming; Yu, Li; Zhang, Chao

doi:10.3389/fmicb.2023.1076947

Cited by 3 publications

(2 citation statements)

References 135 publications

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“…Here, we have developed a method based on dCas9-SSAP and successfully adapted it to Plasmodium yoelii , by effectively achieving the targeted gene deletion and gene tagging, even using relatively shorter DNA templates. It has been estimated that over one-third of the Plasmodium encoding genes remain uncharacterized and this situation is likely not to change if new effective functional characteristics tools are not added ( 17 – 19 ). Our results showed the practicability of dCas9-SSAP in Plasmodium yoelii and thus provided an additional tool to the existing toolbox for elucidating Plasmodium gene functions.…”

Section: Discussionmentioning

confidence: 99%

The dCas9-based genome editing in Plasmodium yoelii

Zhang,

Yang,

Quansah

et al. 2024

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Genetic editing is a powerful tool for functional characterization of genes in various organisms. With its simplicity and specificity, the CRISPR-Cas9 technology has become a popular editing tool, which introduces site-specific DNA double-strand breaks (DSBs), and then leverages the endogenous repair pathway for DSB repair via homology-directed repair (HDR) or the more error-prone non-homologous end joining (NHEJ) pathways. However, in the Plasmodium parasites, the lack of a typical NHEJ pathway selects for DSB repair through the HDR pathway when a homologous DNA template is available. The AT-rich nature of the Plasmodium genome exacerbates this drawback by making it difficult to clone longer homologous repair DNA templates. To circumvent these challenges, we adopted the hybrid catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP) editor to the Plasmodium genome. In Plasmodium yoelii , we demonstrated the use of the dCas9-SSAP, as the cleavage-free gene editor, by targeted gene deletion and gene tagging, even using shorter homologous DNA templates. This dCas9-SSAP method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, would be a great addition to the existing genetic toolbox and could be deployed for the functional characterization of genes in Plasmodium , contributing to improving the ability of the malaria research community in characterizing more than half of genes with unknown functions. IMPORTANCE Malaria caused by Plasmodium parasites infection remains a serious threat to human health, with an estimated 249 million malaria cases and 608,000 deaths worldwide in 2022, according to the latest report from the World Health Organization (WHO). Here, we demonstrated the use of dCas9-single-stranded annealing protein, as the cleavage-free gene editor in Plasmodium yoelii , by targeted deletion and gene tagging, even using shorter homologous DNA templates. This method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, showing the potential significance in greatly improving our ability to elucidate gene functions, would contribute to assisting the malaria research community in deciphering more than half of genes with unknown functions to identify new drug and vaccine targets.

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Section: Discussionmentioning

confidence: 99%

The dCas9-based genome editing in Plasmodium yoelii

Zhang,

Yang,

Quansah

et al. 2024

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“…This allows for greater flexibility and a wider range of targets in platforms using Cas13 compared to those using Cas12 [ 33 ]. Another advantage of Cas13 over Cas12 in nucleic acid platforms is its relatively robust collateral activity, making it a more robust tool for the detection of nucleic acids [ 40 ].…”

Section: Discussionmentioning

confidence: 99%

Detection of Nucleic Acids of the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-Based Assay

Calderón,

Barros,

Fernández-Navarro

et al. 2024

Microorganisms

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Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.

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