RNA-Guided Cas12a- and Cas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke,

Wannaporn Ittiprasert, Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USA.
Chawalit Chatupheeraphat, Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USA.
Victoria H. Mann, Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USA.
Wenhui Li, Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USA.
André Miller, Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USA.
Taiwo Ogunbayo, Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USA.
Kenny Tran, Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USA.
Yousef N. Alrefaei, Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USA.
Margaret Mentink-Kane, Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD 20850, USA.
Paul J. Brindley, Department of Microbiology, Immunology & Tropical Medicine, & Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, DC 20037, USA.

Document Type

Journal Article

Publication Date

1-6-2022

Journal

International journal of molecular sciences

Volume

23

Issue

2

DOI

10.3390/ijms23020631

Keywords

AsCas12a; CRISPR; SpCas9; genome editing; homology directed repair; nonhomologous end-joining; omega-1; ribonucleoprotein complex; schistosome egg

Abstract

The efficiency of the RNA-guided Cas12a nuclease of sp. was compared with Cas9 from , for functional genomics in . 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. Cas12a was more efficient than 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 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 Cas9 plus ssODN, Cas12a plus NT-ssODN, and Cas12a plus T-ssODN groups, respectively. -cleavage against the ssODNs by activated Cas12a was not apparent in vitro. 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 Cas12a induced fewer mutations per genome than Cas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Department

Microbiology, Immunology, and Tropical Medicine

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