PMC

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 ) and enhances the mutagenic DSB-repair via altNHEJ, resulting in more indel mutations at the targeted gene.

(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.

(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.

(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).