Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes

Abstract

TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species.

Figure 1. TALEN constructs and the validation of their activity in rat fibroblasts.

(A) Structure of engineered TALENs binding to exon 2 of rat Tyrosinase (Tyr) gene. (B) TALEN scaffolds (upper) and N- and C-terminal truncated scaffolds (lower), respectively. (C) Relative TALEN activity measured by a single-strand annealing (SSA) assay in human embryonic kidney 293T (HEK293T) cells. (D) Surveyor (Cel-I) nuclease assay for TALEN-induced mutations in Tyr. Arrowheads indicate the expected positions of the digested products. (E) Surveyor assay showing that increased frequency of TALEN-induced mutations by NC truncation and co-expression of Exo1. Data are expressed as means ± SEM (n = 3). *P<0.01 by Student's t-tests. (F–H) Sequence analyses showing increased mutation efficiency by NC truncation and Exo1 expression. Microhomologous sequences adjacent to the breakpoint are underlined for TALEN (F), TALEN-NC (G), and TALEN-NC + Exo1 (H).

Figure 2. Targeted gene disruption by engineered TALENs in rat embryos.

(A) Injection of TALEN-NC with or without Exo1mRNA into rat fertilized eggs. Exo1 increased the efficiency of TALEN-induced mutations in zygotes by ~5×. (B) Microinjection of TALENs mRNA into male pronuclei of a fertilized egg. (C) Surveyor assay on the PCR products shows a TALEN-induced mutation as the digested products (arrowheads) in lane 6, but could not detect a homozygous mutation in lane 2. (D) Sequence analyses of the PCR products showed a 24-bp deletion in the homozygous alleles (eT-NC-Exo1-#2). Microhomologous sequences adjacent to the breakpoint are underlined.

Figure 3. Efficient generation of knockout rats using TALENs with Exo1.

(A) Microinjection of TALENs, TALENs-NC, TALENs from Cellectis, and TALENs-NC with Exo1 into fertilized eggs of agouti DA rats. Coinjection of TALEN-NC mRNA with Exo1 mRNA provided higher mutational efficiency (25%) in pups. (B) The white coat-color of an albino male rat (right) obtained by coinjection of TALEN-NC and Exo1. An agouti male (left) is a littermate. (C) Sequence analyses on founder rats showed a homozygous 29-bp deletion in Tyr (gT-NC-Exo1-#1). Microhomologous sequences adjacent to the breakpoint are underlined.

Figure 4. Schematics of exonucleases (Exo1) functions in double-stranded break (DSB) repair pathways.

Alternative NHEJ (altNHEJ), or microhomology mediated end-joining (MMEJ), generally repairs DSBs in the absence or failure of the classical NHEJ (cNHEJ) pathway. Overexpression of Exo1 induces increased recessions of DNA-ends at the DSB created by TALENs, which inhibits cNHEJ (See Supplementary Fig. S3) and enhances the mutagenic DSB-repair via altNHEJ, resulting in more indel mutations at the targeted gene.