Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice
Open Access
- 26 February 2020
- Vol. 9 (3), 546
- https://doi.org/10.3390/cells9030546
Abstract
Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).”This publication has 34 references indexed in Scilit:
- Simple knockout by electroporation of engineered endonucleases into intact rat embryosScientific Reports, 2014
- A CRISPR view of developmentJournal of Bone and Joint Surgery, 2014
- Development and Applications of CRISPR-Cas9 for Genome EngineeringCell, 2014
- Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineeringScientific Reports, 2014
- Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNAScientific Reports, 2013
- Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonucleaseNucleic Acids Research, 2013
- One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome EngineeringCell, 2013
- Heritable gene targeting in the mouse and rat using a CRISPR-Cas systemNature Biotechnology, 2013
- One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome EngineeringCell, 2013
- Generation of gene-modified mice via Cas9/RNA-mediated gene targetingCell Research, 2013