The dCas9-based genome editing in Plasmodium yoelii
ABSTRACTGenetic 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 endogeno...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2024-03-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/msphere.00095-24 |
| _version_ | 1797221949132242944 |
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| author | Chao Zhang Shijie Yang Elvis Quansah Ziyu Zhang Weiran Da Bingjie Wang |
| author_facet | Chao Zhang Shijie Yang Elvis Quansah Ziyu Zhang Weiran Da Bingjie Wang |
| author_sort | Chao Zhang |
| collection | DOAJ |
| description | ABSTRACTGenetic 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.IMPORTANCEMalaria 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. |
| first_indexed | 2024-04-24T13:13:33Z |
| format | Article |
| id | doaj.art-1339c5f7f5cc40f39a03acf057de153d |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-04-24T13:13:33Z |
| publishDate | 2024-03-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-1339c5f7f5cc40f39a03acf057de153d2024-04-05T01:32:10ZengAmerican Society for MicrobiologymSphere2379-50422024-03-019310.1128/msphere.00095-24The dCas9-based genome editing in Plasmodium yoeliiChao Zhang0Shijie Yang1Elvis Quansah2Ziyu Zhang3Weiran Da4Bingjie Wang5Department of Microbiology and Parasitology, Anhui Key Laboratory of Zoonoses, School of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaThe Second Clinical Medical College, Anhui Medical University, Hefei, ChinaDepartment of Microbiology and Parasitology, Anhui Key Laboratory of Zoonoses, School of Basic Medical Sciences, Anhui Medical University, Hefei, ChinaThe First Clinical Medical College, Anhui Medical University, Hefei, ChinaThe First Clinical Medical College, Anhui Medical University, Hefei, ChinaThe First Clinical Medical College, Anhui Medical University, Hefei, ChinaABSTRACTGenetic 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.IMPORTANCEMalaria 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.https://journals.asm.org/doi/10.1128/msphere.00095-24malariaPlasmodium parasitesCRISPR-Cas9microbial single-strand annealing proteinsdCas9-SSAP |
| spellingShingle | Chao Zhang Shijie Yang Elvis Quansah Ziyu Zhang Weiran Da Bingjie Wang The dCas9-based genome editing in Plasmodium yoelii mSphere malaria Plasmodium parasites CRISPR-Cas9 microbial single-strand annealing proteins dCas9-SSAP |
| title | The dCas9-based genome editing in Plasmodium yoelii |
| title_full | The dCas9-based genome editing in Plasmodium yoelii |
| title_fullStr | The dCas9-based genome editing in Plasmodium yoelii |
| title_full_unstemmed | The dCas9-based genome editing in Plasmodium yoelii |
| title_short | The dCas9-based genome editing in Plasmodium yoelii |
| title_sort | dcas9 based genome editing in plasmodium yoelii |
| topic | malaria Plasmodium parasites CRISPR-Cas9 microbial single-strand annealing proteins dCas9-SSAP |
| url | https://journals.asm.org/doi/10.1128/msphere.00095-24 |
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