Generation of multi-gene knockout rabbits using the Cas9/gRNA system

Abstract

The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.

Keywords: Cas9/gRNA system; Multiple-gene knockout; Rabbits.

Figure 1

Genome editing via the Cas9/gRNA system in rabbit. (A) Constructs and schematic illustration of the Cas9/gRNA system used in this study. The T7 promoter drives transcription of the gRNA, consisting of a target sequence and a scaffold sequence. NLS: nuclear localization signal. bGH polyA: bovine growth hormone poly-A. (B) Target sequence of the IL2rg, RAG1, RAG2, TIKI1 and ALB genes using the Cas9/gRNA system. gRNA-targeting sequences are underlined and PAM sequences are highlighted in red.

Figure 2

One gene (IL2rg)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the IL2rg gene in injected embryos. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink. (B) Generation of IL2rg KO rabbits via the Cas9/gRNA system. Zygotes (n = 66) microinjected with 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for IL2rg were transferred to 5 recipient mothers, 3 of which gave birth to 8 live kits. (C) Detailed mutations of the IL2rg gene in the 8 KO founders. The number of founder KO rabbits is shown in the left column. (D) Picture of 26 days old IL2rg KO rabbits. (E) The thymus of IL2rg KO rabbits was obviously smaller than that from age-matched WT ones. In A and C, the WT sequence is shown at the top with the target sites in underline; the sizes of the deletions (-) or insertions (+) are shown in the right column. At least 8 TA-clones for each embryos or KO rabbit were used for sequencing to obtain detailed information of the mutation.

Figure 3

One gene (TIKI1)-KO rabbit embryos and newborn rabbits. (A) Sequenced mutations in the TIKI1 gene in injected embryos. Deletions are indicated by dashes and insertions are indicated in blue. (B) Generation of TIKI1 KO rabbits by the Cas9/gRNA system. The zygotes (n = 30) microinjection with the 200 ng/μL of Cas9 mRNA and 20 ng/μL of gRNA for TIKI1 were transferred to 3 recipient mothers, 2 of which gave birth to 5 live kits. (C) Detailed mutations of the TIKI1 gene in the 5 KO founders. The number of founder KO rabbits is shown in the left column. In A and C, the WT sequence is shown at the top with the target sites in underline, and the sizes of the deletions (-) or insertions (+) are shown in the right column.

Figure 4

Two gene-KO rabbits obtained by the Cas9/gRNA system in a single step. (A) Generation of 2 gene-KO (IL2rg/RAG1) rabbits via the Cas9/gRNA system. Zygotes (n = 67) were microinjected with 200 ng/μL of Cas9 mRNA, 20 ng/μL of gRNA for IL2rg and 20 ng/μL of gRNA for RAG1 and transferred into 5 recipient mothers, 2 of which gave birth to 5 live kits. (B) Detailed mutations of the IL2rg and RAG1 genes in the 5 KO founders. The number of founder KO rabbits is shown in the left column. For each gene, the WT sequence is shown at the top with the target sites in underline, deletions are indicated by dashes and insertions are indicated in blue, and the sizes of the deletions (-) or insertions (+) are shown in the right column.

Figure 5

Off-target analysis of Cas9/gRNA system-induced mutations in KO founders. (A) T7EI assays of the PCR products of candidate off-target sites using the pooled DNA of all KO founders for each gene as the template (primer sequences listed in Additional file 3: Table S2). The IOT5 could be cleaved (red arrowheads). (B) Sequencing diagram of IOT5 in #1 IL2rg KO new-born rabbits showing a double curve after the mutation around the PAM region. (C) Detailed mutations of IOT5 in the newborn rabbits. The number of founder KO rabbits is shown in the left column. The WT sequence is shown at the top with the target sites in underline. Deletions are indicated by dashes, insertions are indicated in blue and substitutions are indicated in pink, and the sizes of the deletions (-) or insertions (+) are shown in the right column. The fractions indicate the read number of the mutant allele (numerator) out of total read number (denominator).