Kyoungmi Kim

Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins.

807 To the Editor: Cpf1 represents a class 2/type V CRISPR RNA-guided endonuclease1 that is distinct from the type II CRISPR-Cas9 nuclease, widely used for genome editing2–5. It recognizes thymidine-rich protospaceradjacent motif (PAM) sequences, expanding the range of RNA-guided genome editing beyond guanosine-rich PAM sequences recognized by Streptococcus pyogenes Cas9 (SpCas9). Yet, the potential of Cpf1 for targeted mutagenesis in whole organisms has not been demonstrated. Here, we report generation of mutant mice using preassembled recombinant CRISPR-Cpf1 ribonucleoproteins (RNPs). First, we expressed Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) in Escherichia coli and purified the resulting recombinant Cpf1 protein (Supplementary Table 1 and Supplementary Methods). We tested the activity of the purified Cpf1 protein in vitro (Supplementary Fig. 1a) and in mouse NIH3T3 cells. Preassembled Cpf1 RNPs cleaved target DNA efficiently in vitro and induced small insertions and deletions (indels) in NIH3T3 cells, with frequencies of up to 45% (Supplementary Fig. 1b), at the target sites in Foxn1 (Fig. 1a), which encodes Forkhead box protein N1, a transcription factor. Having confirmed that Cpf1 RNPs were highly active in vitro and in cultured cells, we carried out Cpf1 RNP-mediated genome editing in mouse embryos. We microinjected the AsCpf1 RNPs into 36 one-cell-stage embryos, cultured them in vitro, and obtained 12 blastocysts (Supplementary Fig. 2a). T7 endonuclease 1 (T7E1) assay and targeted deep sequencing showed that 10 out of 12 (83%) blastocysts contained Cpf1-mediated mutations in Foxn1 (Supplementary Fig. 2b,c and Supplementary Table 2,3). SpCas9 RNPs targeted to the same region in the gene showed a comparable efficiency (90%) (Supplementary Fig. 3). No off-target mutations were detected at homologous sites with up to 4-nucleotide mismatches using the T7E1 assay and targeted deep sequencing (Supplementary Table 4 and Supplementary Fig. 4), suggesting that Cpf1 RNPs were highly specific. Next, we investigated whether both Cas9 RNPs and Cpf1 RNPs could be delivered into animal embryos by electroporation rather than microinjection. Although Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins

Tumor-specific apoptosis caused by deletion of the ERBB3 pseudo-kinase in mouse intestinal epithelium

Pharmacologic blockade of EGFR or the closely related receptor ERBB2 has modest efficacy against colorectal cancers in the clinic. Although the upregulation of ERBB3, a pseudo-kinase member of the EGFR/ERBB family, is known to contribute to EGFR inhibitor resistance in other cancers, its functions in normal and malignant intestinal epithelium have not been defined. We have shown here that the intestinal epithelium of mice with intestine-specific genetic ablation of Erbb3 exhibits no cytological abnormalities but does exhibit loss of expression of ERBB4 and sensitivity to intestinal damage. By contrast, intestine-specific Erbb3 ablation resulted in almost complete absence of intestinal tumors in the ApcMin mouse model of colon cancer. Unlike nontransformed epithelium lacking ERBB3, intestinal tumors lacking ERBB3 had reduced PI3K/AKT signaling, which led to attenuation of tumorigenesis via a tumor-specific increase in caspase-3-mediated apoptosis. Consistent with the mouse data, which suggest that ERBB3-ERBB4 heterodimers contribute to colon cancer survival, experimentally induced loss of ERBB3 in a KRAS mutant human colon cancer cell line was associated with loss of ERBB4 expression, and siRNA knockdown of either ERBB3 or ERBB4 resulted in elevated levels of apoptosis. These results indicate that the ERBB3 pseudo-kinase has essential roles in supporting intestinal tumorigenesis and suggest that ERBB3 may be a promising target for the treatment of colorectal cancers.

Epiregulin-dependent amphiregulin expression and ERBB2 signaling are involved in luteinizing hormone-induced paracrine signaling pathways in mouse ovary.

Sustained EGF receptor (EGFR) phosphorylation by de novo synthesis of EGFR ligands plays an essential role in mediating luteinizing hormone (LH)-induced ovulation process in the preovulatory follicles (POFs). In the present study, the effect of epiregulin (EREG) on oocyte maturation and ovulation was investigated using Ereg knockout (Ereg-/-) mice congenic on a C57BL/6 background. Rate of spontaneous oocyte meiotic resumption of denuded oocytes (DOs) or cumulus cell-oocyte complexes (COCs) in vitro is similar between wild-type and Ereg-/- mice. However, gonadotropin-induced meiotic resumption in vivo is attenuated, and the number of COCs with expanded cumulus matrix and superovulated eggs dramatically decrease in Ereg-/- mice. Nonetheless, the number of eggs ovulated during normal estrus cycles and litter sizes in Ereg-/- mice are comparable to those of wild-type littermates. In contrast to other EGFR ligands, induction of amphiregulin (Areg) mRNA is severely reduced in ovaries collected from Ereg-/- mice either after human chorionic gonadotropin (hCG) treatment in immature mice or LH surge in adults. Gonadotropin-induced EGFR and ERBB2 phosphorylation in ovaries is attenuated in immature Ereg-/- mice, and MAPK3/1 phosphorylation and prostaglandin synthase 2 (PTGS2) protein levels are reduced. This attenuation, however, is no longer detectable in adult Ereg-/- mice after LH surge. This study implicates that EREG mediates signals downstream of Areg mRNA expression and that EGFR-ERBB2 signals contributes to regulation of ovulation process.

Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy

Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.

Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26(tm1Sor), revealed that treatment of 1-cell or 2-cell embryos with 3microM of Cre fusion protein for 2h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

Questioning unexpected CRISPR off-target mutations in vivo

In summary, the CRISPR-Cas9-treated mice do not exhibit more variants that are absent in the uncorrected control than vice versa. Furthermore, the observed variants are extremely unlikely a consequence of a mutagenic process in the investigated animal, but are much better explained by differences in the genetic background between the two gene-edited mice and the control mouse. We conclude that the data presented by Schaefer et al does not provide any evidence for off-target mutagenesis due to CRISPR-Cas9 treatment and see no reason to be doubtful about the previous findings that Cas9 does not cause SNVs and indels at non-homologous, PAM-free sites.

Arhgap17, a RhoGTPase activating protein, regulates mucosal and epithelial barrier function in the mouse colon.

Coordinated regulation of the actin cytoskeleton by the Rho GTPase family is required for the maintenance of polarity in epithelial cells as well as for their proliferation and migration. A RhoGTPase-activating protein 17 (Arhgap17) is known to be involved in multiple cellular processes in vitro, including the maintenance of tight junctions and vesicle trafficking. However, the function of Arhgap17 has not been studied in the physiological context. Here, we generated Arhgap17-deficient mice and examined the effect in the epithelial and mucosal barriers of the intestine. Reporter staining revealed that Arhgap17 expression is limited to the luminal epithelium of intestine. Arhgap17-deficient mice show an increased paracellular permeability and aberrant localization of the apical junction complex in the luminal epithelium, but do not develop spontaneous colitis. The inner mucus layer is impervious to the enteric bacteria irrespective of Tff3 downregulation in the Arhgap17-deficient mice. Interestingly however, treatment with dextran sulfate sodium (DSS) causes an increased accumulation of DSS and TNF production in intraluminal cells and rapid destruction of the inner mucus layer, resulting in increased severity of colitis in mutant mice. Overall, these data reveal that Arhgap17 has a novel function in regulating transcellular transport and maintaining integrity of intestinal barriers.