Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering

Abstract

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.

Figure 1. CRISPR/Cas9-mediated knock-in strategy using the hsp70 promoter.

(A) For the generation of knock-in transgenic fish, sgRNA1 (for genome digestion), sgRNA2 (for plasmid digestion), the donor plasmid having a bait sequence, and Cas9 mRNA are co-injected into one-cell stage zebrafish embryos. (B) After injection, CRISPR/Cas9-mediated cleavage occurs in the genome at the site upstream (approximately, 200–600 bp) of the gene of interest (gene X). CRISPR/Cas9-mediated cleavage also occurs in the donor plasmid at the bait sequence. This leads to a homology independent DNA repair, resulting in the integration of the donor plasmid into the targeted locus. Both forward and reverse integrations occur. Cis-regulatory DNA sequences for geneX expression act on the hsp70 promoter (enhancer-trapping), resulting in the expression of the reporter gene in cells that express gene X.

Figure 2. Generation of evx2-hs:Gal4 and eng1b-hs:Gal4 transgenic fish.

(A) A schematic of the donor plasmid, Gbait-hs-Gal4. The plasmid consists of Gbait (a bait sequence derived from GFP15), hsp70 promoter (hsP), Gal4, and polyA (pA). (B) A 3-dpf Tg[UAS:RFP] embryo showing RFP expression after co-injection of evx2sg3, sgG, the donor plasmid, and Cas9 mRNA. (C) A 3-dpf Tg[evx2-hs:Gal4]; Tg[UAS:GFP] embryo. (D) Spinal cord of a 1.4-dpf Tg[eng1b-hs:Gal4]; Tg[UAS:GFP] embryo was stained with an anti-Evx2 antibody. GFP and Evx2 signals broadly overlap, although some of GFP cells do not have a detectable level of Evx2 signal. (E) A 3-dpf Tg[UAS:RFP] embryo showing RFP expression after co-injection of eng1bsg5, sgG, the donor plasmid, and Cas9 mRNA. RFP is expressed in a subset of neurons in the CNS, cells at the midbrain-hindbrain boundary (MHB), muscle cells possibly derived from muscle pioneers (MP). (F), A 3-dpf Tg[eng1b-hs:Gal4]; Tg[UAS:GFP] embryo. (G) Spinal cord of a 1.4-dpf Tg[eng1b-hs:Gal4]; Tg[UAS:GFP] embryo was stained with an anti-En1 antibody. GFP and En1 signals almost completely overlap. Scale bars: 200 μm in B, C, E, and F; 20 μm in D and G.

Figure 3. Generation of lRl-GFPTx and lRl-ChR transgenic fish for glyt2, vglut2a, and eng1b loci.

(A) Schematics of the donor plasmids having the lRl (loxP-RFP-loxP) expression cassette. The Tbait-hs-lRl-GFPTx and Mbait-hs-lRl-GFPTx plasmids consist of Tbait (a bait sequence derived from Tet113) or Mbait (a bait sequence derived from Mc4r18), hsp70 promoter (hsP), lRl, GFPTx (a fusion construct of GFP and Tetanus-toxin light chain, and polyA (pA). In the Mbait-hs-lRl-GFPTx-truncate plasmid, a part of Tetanus-toxin light chain and polyA are removed from the Mbait-hs-lRl-GFPTx. The Mbait-hs-lRl-ChR has a ChR-YFP fusion construct in the position of GFPTx. (B) A 3-dpf embryo showing RFP expression after co-injection of glyt2sg2, sgM, Mbait-hs-lRl-GFPTx plasmid, and Cas9 mRNA. (C) A 3-dpf Tg[glyt2-hs:lRl-GFPTx] embryo. (D) Spinal cord of a Tg[glyt2-hs:lRl-GFPTx]; Tg[BAC-vglut2a-hs:GFP] embryo at 3 dpf. RFP-positive cells (prospective glycinergic neurons) and GFP-positive cells (prospective glutamatergic neurons) are mutually exclusive. (E) Spinal cord of a Tg[glyt2-hs:lRl-GFPTx]; Tg[BAC-dbx1b-hs:Cre] embryo at 3 dpf. GFPTx is expressed in cells that are likely derived from dbx1b-positive cells. (F) Spinal cord of a Tg[glyt2-hs:lRl-ChR]; Tg[BAC-dbx1b-hs:Cre] embryo at 3 dpf. ChR-YFP is expressed in cells that are likely derived from dbx1b-positive cells. (G) A 3-dpf Tg[vglut2a-hs:lRl-GFPTx] embryo. (H) Spinal cord of a Tg[vglut2a-hs:lRl-GFPTx]; Tg[BAC-vglut2a-hs:GFP] embryo at 3dpf. RFP and GFP cells almost completely overlap. (I) A 3-dpf Tg[eng1b-hs:lRl-GFPTx] embryo. Scale bars: 200 μm in B, C, G, and I; 20 μm in D, E, F and H.