Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum
Summary: It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present a genetic screening tool to assess the contribution of essential genes from Plasmodium falciparum by the conditional CRISPR-/deadCas9-based interference and activ...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2024-04-01
|
| Series: | iScience |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589004224008241 |
| _version_ | 1797220289952612352 |
|---|---|
| author | Amuza Byaruhanga Lucky Chengqi Wang Xiaolian Li Xiaoying Liang Azhar Muneer Jun Miao |
| author_facet | Amuza Byaruhanga Lucky Chengqi Wang Xiaolian Li Xiaoying Liang Azhar Muneer Jun Miao |
| author_sort | Amuza Byaruhanga Lucky |
| collection | DOAJ |
| description | Summary: It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present a genetic screening tool to assess the contribution of essential genes from Plasmodium falciparum by the conditional CRISPR-/deadCas9-based interference and activation (i/a) systems. We screened both CRISPRi and CRISPRa sets, consisting of nine parasite lines per set targeting nine genes via their respective gRNAs. By conducting amplicon sequencing of gRNA loci, we identified the contribution of each targeted gene to parasite fitness upon drug (artemisinin, chloroquine) and stress (starvation, heat shock) treatment. The screening was highly reproducible, and the screening libraries were easily generated by transfection of mixed plasmids expressing different gRNAs. We demonstrated that this screening is straightforward, robust, and can provide a fast and efficient tool to study essential genes that have long presented a bottleneck in assessing their functions using existing genetic tools. |
| first_indexed | 2024-04-24T12:47:11Z |
| format | Article |
| id | doaj.art-bfa4d16cc4b84e758e80338abfbe8415 |
| institution | Directory Open Access Journal |
| issn | 2589-0042 |
| language | English |
| last_indexed | 2024-04-24T12:47:11Z |
| publishDate | 2024-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj.art-bfa4d16cc4b84e758e80338abfbe84152024-04-07T04:36:08ZengElsevieriScience2589-00422024-04-01274109602Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparumAmuza Byaruhanga Lucky0Chengqi Wang1Xiaolian Li2Xiaoying Liang3Azhar Muneer4Jun Miao5Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USACenter for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USADepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USADepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USADepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USADepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA; Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA; Corresponding authorSummary: It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present a genetic screening tool to assess the contribution of essential genes from Plasmodium falciparum by the conditional CRISPR-/deadCas9-based interference and activation (i/a) systems. We screened both CRISPRi and CRISPRa sets, consisting of nine parasite lines per set targeting nine genes via their respective gRNAs. By conducting amplicon sequencing of gRNA loci, we identified the contribution of each targeted gene to parasite fitness upon drug (artemisinin, chloroquine) and stress (starvation, heat shock) treatment. The screening was highly reproducible, and the screening libraries were easily generated by transfection of mixed plasmids expressing different gRNAs. We demonstrated that this screening is straightforward, robust, and can provide a fast and efficient tool to study essential genes that have long presented a bottleneck in assessing their functions using existing genetic tools.http://www.sciencedirect.com/science/article/pii/S2589004224008241GeneticsTechniques in geneticsParasitologyGenomic analysis |
| spellingShingle | Amuza Byaruhanga Lucky Chengqi Wang Xiaolian Li Xiaoying Liang Azhar Muneer Jun Miao Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum iScience Genetics Techniques in genetics Parasitology Genomic analysis |
| title | Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum |
| title_full | Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum |
| title_fullStr | Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum |
| title_full_unstemmed | Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum |
| title_short | Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum |
| title_sort | transforming the crispr dcas9 based gene regulation technique into a forward screening tool in plasmodium falciparum |
| topic | Genetics Techniques in genetics Parasitology Genomic analysis |
| url | http://www.sciencedirect.com/science/article/pii/S2589004224008241 |
| work_keys_str_mv | AT amuzabyaruhangalucky transformingthecrisprdcas9basedgeneregulationtechniqueintoaforwardscreeningtoolinplasmodiumfalciparum AT chengqiwang transformingthecrisprdcas9basedgeneregulationtechniqueintoaforwardscreeningtoolinplasmodiumfalciparum AT xiaolianli transformingthecrisprdcas9basedgeneregulationtechniqueintoaforwardscreeningtoolinplasmodiumfalciparum AT xiaoyingliang transformingthecrisprdcas9basedgeneregulationtechniqueintoaforwardscreeningtoolinplasmodiumfalciparum AT azharmuneer transformingthecrisprdcas9basedgeneregulationtechniqueintoaforwardscreeningtoolinplasmodiumfalciparum AT junmiao transformingthecrisprdcas9basedgeneregulationtechniqueintoaforwardscreeningtoolinplasmodiumfalciparum |