Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

Identification and functional characterization of a novel MUTYH gene mutation.

e12026 Background: Biallelic germline mutations in MUTYH result in the autosomal recessive syndrome of MUTYH associated polyposis (MAP).Three well-known, common mutations account for the vast majority of identifiable germline mutations, and serve as the basis for current genetic testing strategies. Comprehensive sequencing of MUTYH often identifies variants of uncertain pathologic significance, and studies to determine the pathogenicity of newly identified variants may offer valuable clinical information and mechanistic insights. In the present study we seek to describe the base-excision repair function of a novel MUTYH (p.C306W) mutation identified in a patient with multiple colon polyps and a family history of colon cancers. Methods: A 50 year old patient with >50 adenomas underwent clinical and laboratory evaluation to assess for germline genetic mutations. We performed Sanger sequencing of tumor and germline DNA together with targeted restriction enzyme digest of germline DNA and fragment DNA sequencing of the alleles. We prepared MUTYH proteins with protein liquid chromatography and assessed their mismatched adenine excision repair capacity employing a glycosylase assay. Results: Analysis of the patient's germline DNA revealed an absence of APC mutations, and the presence of the previously well characterized p.G396D MUTYH mutation as well as a novel p.C306W mutation. Targeted restriction enzyme digest demonstrated trans configuration of the p.G396D and p.C306W MUTYH mutations. Mismatched adenine excision functionality of wildtype MUTYH, known mutant controls p.G396D and p.Y179C, and putative mutant p.C306W were assessed in the glycosylase assay. Consistent with previous experimental observations, relative to wildtype MUTYH, the p.G396D and p.Y179C MUTYH mutants demonstrated attenuated adenine excision activities of 43% and 0%, respectively. Comparable to the activity of the pY179C mutant, the novel p.C306D mutant demonstrated 0% adenine excision activity. Subsequent tumor analysis demonstrated G:C to T:A transversion in the APC gene in somatic DNA derived from an adenoma. Conclusions: Experimental and clinical data demonstrate that p.C306D MUTYH is a pathogenic mutation contributing to the phenotype of MAP.

Antimicrobial Susceptibilities of Salmonella Isolates Obtained from Layer Chicken Houses on a Commercial Egg-Producing Farm in Japan, 1997 to 2002

Susceptibility to antimicrobial agents was examined for 325 isolates of Salmonella enterica serotypes Cerro, Infantis, Livingstone, and Montevideo isolated from layer houses on a commercial egg-production farm in the western region of Japan between 1997 and 2002. No antimicrobials were used for therapeutic purposes on the farm during this period. From 1.8 to 3.1% of the isolates were resistant to ampicillin, chloramphenicol, and tetracycline. Resistance to streptomycin and sulfisoxazole was found in 52.9 and 65.5%, respectively, of Salmonella Montevideo isolates and in 0 to 13.2% of the isolates of the other serotypes. All the streptomycin-resistant isolates of Salmonella Montevideo also exhibited resistance to sulfisoxazole. Salmonella Montevideo isolates were first isolated in 1998, and 80.0% of the isolates obtained in this year were resistant to streptomycin and sulfisoxazole. The results suggest that Salmonella Montevideo isolates that had already acquired resistance may have been introduced into the layer houses, although the route and vehicle of transmission were uncertain. The proportion of Salmonella Montevideo isolates resistant to streptomycin and sulfisoxazole significantly decreased (P < 0.01) from 79.5% for 1998 through 1999 to 37.3% for 2000 through 2002. This decrease probably was due to the fact that no antimicrobials were used on the farm. Among 10 isolates from different serotypes obtained from 1997 to 2002 that were resistant to ampicillin, two and five isolates harbored 42- and 63-kb R plasmids and identical DraI restriction enzyme digest patterns, respectively, and carried the blaTEM gene. The results suggest that the ampicillin resistance determinants were transferred among different serotypes of Salmonella in the layer houses.