Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.

Figure 1

Creation of mutant mice with a 2-Mb deletion with a donor plasmid. (A) Schematic representation of the 2-Mb region upstream of Sox9. The black line represents genome sequence. Homology arms are indicated with black and gray boxes. PCR primers are indicated with arrows. Positions of sgRNA recognition sequences are indicated with scissors. (B) An agarose gel electrophoretogram image of PCR products amplified using 2F1 and 1R1 primers and DNA prepared from #48 embryo. M: a 1-kb DNA ladder (molecular weight markers); N: no template control. (C) Sequence of the deletion junction. Sequences of the donor plasmid and of #48 embryo are aligned. Positions corresponding to arms and the ClaI recognition sequence are indicated at the top. Parts of gRNA recognition sequences are underlined. Black and gray boxes correspond to those of (A).

Figure 2

Generation of mutant mice with a 5-Mb deletion with or without a donor plasmid. (A) Schematic representation of the 5-Mb region upstream of Sox9. The black line represents a genome sequence. Homology arms in a donor plasmid are indicated with black and gray boxes. PCR primers are indicated with arrows. Positions of sgRNA recognition sequences are denoted with scissors. (B) An agarose gel electrophoretogram image of PCR products amplified using 3F1 and 1R1 primers and DNA prepared from embryos injected with the donor plasmid (left panel) and without the donor plasmid (right panel). Embryo IDs are shown above images with the # sign. M: a 1-kb DNA ladder (markers); N: no template control. (C) Sequences of the donor plasmid and of embryos injected with the donor plasmid are aligned. Positions corresponding to arms and ClaI recognition sequence are indicated at the top. (D) Sequences of the donor plasmid and of embryos injected without the donor plasmid are aligned. Positions corresponding to arms and ClaI recognition sequence are indicated at the top. Parts of gRNA recognition sequences are underlined. Black and gray boxes correspond to those in (A).

Figure 3

Generation of mutant mice with a 5-Mb deletion using an ssODN template. (A) Schematic representation of the 5-Mb region upstream of Sox9. The black line represents genome sequence. Homology arms in the ssODN are indicated with black and gray boxes. PCR primers are indicated with arrows. Positions of sgRNA recognition sequences are denoted with scissors. (B) An agarose gel electrophoretogram image of PCR products amplified using 3F1 and 1R1 primers and DNA prepared from embryos injected with the ssODN template. Embryo IDs are shown above images with the # sign. M: a 1-kb DNA ladder (markers); N: no template control. (C) Sequences of the ssODN template and of embryos injected with the ssODN template are aligned. Positions corresponding to arms and ClaI recognition sequence are indicated at the top. Parts of gRNA recognition sequences are underlined. The asterisk indicates an unknown sequence. The obelisk indicates genomic sequence nonhomologous to the ssODN or to the internal sequence flanked by sgRNA3 and sgRNA1. Black and gray arrows correspond to those in (A).

Figure 4

Generation of mutant mice with a 5-Mb inversion. (A) Schematic representation of the 5-Mb region upstream of Sox9. The black line represents a genome sequence. Homology arms are indicated with black (arm1) and gray (arm3) boxes. PCR primers are indicated with arrows. The inverted region is denoted by a box containing arrowheads. (B) Agarose gel electrophoretogram images of PCR products amplified using 3F2/1F1 (upper panel), 3R1/1R2 (middle panel), 3F3/1F2 (lower left panel), and 3R3/1R4 (lower right panel) primer sets and DNA prepared from the embryos. Sample ID is indicated at top of each panel. Arrows and arrowheads indicate positive and nonspecific signals, respectively. M: a 100-bp DNA ladder (markers); N: no-template control. (C) Sequences of inversion junctions. Results of direct sequencing of PCR products shown in (B). Corresponding sequences of arms and inverted sequences are shown at the top of each panel. Parts of gRNA recognition sequences are underlined. Asterisks indicate an unknown sequence. The obelisk indicates a genomic sequence nonhomologous to the arms or to an internal sequence flanked by sgRNA3 and sgRNA1. Black and gray arrows correspond to those in (A). The inverted region is indicated with a box containing arrowheads as shown in (A).

Figure 5

FISH analysis of 2-Mb and 5-Mb deletions. (A) Schematic representation of the positions of BAC probes. BACs located within the sgRNAs are shown with green lines and letters, and those outside in red lines and letters. (B,C) Representative nucleus images of DNA FISH analysis of fibroblasts obtained from #48 embryo of Fig. 1 (B, 2-Mb deletion) and #13 embryo of Fig. 2 (C, 5-Mb deletion). Probes that we used are indicated at the top of the images with probe colors. Enlarged images of boxed areas are placed on the right.