Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis.
CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.
Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell biology, Agency of Science, Technology and Research (A-STAR), Singapore.
Raymond Teck Ho
http://orcid.org/0000-0001-6522-3043
RT
Lee
Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell biology, Agency of Science, Technology and Research (A-STAR), Singapore.
Ashley Shu Mei
AS
Ng
Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell biology, Agency of Science, Technology and Research (A-STAR), Singapore.|Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
Philip W
PW
Ingham
D000818:Animals; D064113:CRISPR-Cas Systems; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D016337:RNA, Catalytic; D000094704:RNA, Guide, CRISPR-Cas Systems