RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i>
The efficiency of the RNA-guided <i>As</i>Cas12a nuclease of <i>Acidaminococcus</i> sp. was compared with <i>Sp</i>Cas9 from <i>Streptococcus pyogenes</i>, for functional genomics in <i>Schistosoma mansoni</i>. We deployed optimized conditi...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/2/631 |
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| author | Wannaporn Ittiprasert Chawalit Chatupheeraphat Victoria H. Mann Wenhui Li André Miller Taiwo Ogunbayo Kenny Tran Yousef N. Alrefaei Margaret Mentink-Kane Paul J. Brindley |
| author_facet | Wannaporn Ittiprasert Chawalit Chatupheeraphat Victoria H. Mann Wenhui Li André Miller Taiwo Ogunbayo Kenny Tran Yousef N. Alrefaei Margaret Mentink-Kane Paul J. Brindley |
| author_sort | Wannaporn Ittiprasert |
| collection | DOAJ |
| description | The efficiency of the RNA-guided <i>As</i>Cas12a nuclease of <i>Acidaminococcus</i> sp. was compared with <i>Sp</i>Cas9 from <i>Streptococcus pyogenes</i>, for functional genomics in <i>Schistosoma mansoni</i>. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. <i>As</i>Cas12a was more efficient than <i>Sp</i>Cas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With <i>As</i>Cas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the <i>Sp</i>Cas9 plus ssODN, <i>As</i>Cas12a plus NT-ssODN, and <i>As</i>Cas12a plus T-ssODN groups, respectively. <i>Trans</i>-cleavage against the ssODNs by activated <i>As</i>Cas12a was not apparent in vitro. <i>Sp</i>Cas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although <i>As</i>Cas12a induced fewer mutations per genome than <i>Sp</i>Cas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases. |
| first_indexed | 2024-03-10T01:19:46Z |
| format | Article |
| id | doaj.art-25e7ca30079d40aaacb0c3da214e9942 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T01:19:46Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-25e7ca30079d40aaacb0c3da214e99422023-11-23T14:01:35ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-01-0123263110.3390/ijms23020631RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i>Wannaporn Ittiprasert0Chawalit Chatupheeraphat1Victoria H. Mann2Wenhui Li3André Miller4Taiwo Ogunbayo5Kenny Tran6Yousef N. Alrefaei7Margaret Mentink-Kane8Paul J. Brindley9Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USADepartment of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USADepartment of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USADepartment of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USASchistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USASchistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USASchistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USADepartment of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USASchistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USADepartment of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USAThe efficiency of the RNA-guided <i>As</i>Cas12a nuclease of <i>Acidaminococcus</i> sp. was compared with <i>Sp</i>Cas9 from <i>Streptococcus pyogenes</i>, for functional genomics in <i>Schistosoma mansoni</i>. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. <i>As</i>Cas12a was more efficient than <i>Sp</i>Cas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With <i>As</i>Cas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the <i>Sp</i>Cas9 plus ssODN, <i>As</i>Cas12a plus NT-ssODN, and <i>As</i>Cas12a plus T-ssODN groups, respectively. <i>Trans</i>-cleavage against the ssODNs by activated <i>As</i>Cas12a was not apparent in vitro. <i>Sp</i>Cas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although <i>As</i>Cas12a induced fewer mutations per genome than <i>Sp</i>Cas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.https://www.mdpi.com/1422-0067/23/2/631<i>As</i>Cas12a<i>Sp</i>Cas9ribonucleoprotein complexCRISPRomega-1genome editing |
| spellingShingle | Wannaporn Ittiprasert Chawalit Chatupheeraphat Victoria H. Mann Wenhui Li André Miller Taiwo Ogunbayo Kenny Tran Yousef N. Alrefaei Margaret Mentink-Kane Paul J. Brindley RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i> International Journal of Molecular Sciences <i>As</i>Cas12a <i>Sp</i>Cas9 ribonucleoprotein complex CRISPR omega-1 genome editing |
| title | RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i> |
| title_full | RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i> |
| title_fullStr | RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i> |
| title_full_unstemmed | RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i> |
| title_short | RNA-Guided <i>As</i>Cas12a- and <i>Sp</i>Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, <i>Schistosoma mansoni</i> |
| title_sort | rna guided i as i cas12a and i sp i cas9 catalyzed knockout and homology directed repair of the omega 1 locus of the human blood fluke i schistosoma mansoni i |
| topic | <i>As</i>Cas12a <i>Sp</i>Cas9 ribonucleoprotein complex CRISPR omega-1 genome editing |
| url | https://www.mdpi.com/1422-0067/23/2/631 |
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