Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old-new

Abstract

In the recent years, sequence-specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionzed the fields of animal genome editing and transgenesis. However, these new techniques require microinjection to deliver nucleic acids into embryos to generate gene-modified animals. Microinjection is a delicate procedure that requires sophisticated equipment and highly trained and experienced technicians. Though over a dozen alternate approaches for nucleic acid delivery into embryos were attempted during the pre-CRISPR era, none of them became routinely used as microinjection. The addition of CRISPR/Cas9 to the genome editing toolbox has propelled the search for novel delivery approaches that can obviate the need for microinjection. Indeed, some groups have recently developed electroporation-based methods that have the potential to radically change animal transgenesis. This review provides an overview of the old and new delivery methods, and discusses various strategies that were attempted during the last three decades. In addition, several of the methods are re-evaluated with respect to their suitability to deliver genome editing components, particularly CRISPR/Cas9, to embryos.

Keywords: CRISPR/Cas; Genome editing; Mcroinjection; Nucleic acids delivery; Pronuclaer injection; Transgenic.

Fig. 1

Schematic showing various Knoc-kout/Knock-In (KO/KI) and Transgenic (TG) mice generation approaches reviewed in the article. Only the methods that have potential for generating germ-line transmitted offspring are shown (see text for additional methods that may not have germ-line transmission potential). (A) The classical methods that require microinjection are shown in inner circle, [*; the CRISPR system can generate KO/KI models directly through PI and therefore can by-pass the use of ES cells]. (B) The approaches that do not require microinjection are listed in the middle circle (those in the dark gray shaded area do not require ex vivo handling of embryos). (C) The GONAD method that does not require both microinjection- and ex vivo handling- of embryos, is listed in the outer circle

Fig. 2

Ex vivo delivery methods to pre-implantation embryos and sperm

Fig. 3

In vivo delivery to pre-implantation embryos, fetuses and ovarian tissues

Fig. 4

In vivo delivery to male gonadal tissues