http://knockout.cwru.edu/publications/hsucrisprreview.pdf

Development and applications of CRISPR-Cas9 for genome engineering.

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

Development and Applications of CRISPR-Cas9 for Genome Engineering

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(13)00087-5.pdf

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

http://stacks.cdc.gov/view/cdc/29957/cdc_29957_DS1.pdf

CRISPR-Cas systems for editing, regulating and targeting genomes

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene

/pdf/efficient-design-and-assembly-of-custom-talen-and-other-tal-2x34j59pst.pdf

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=1117&context=gdcb_las_pubs

Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

The aim of gene therapy for cystic fibrosis (CF) lung disease is to efficiently and safely express the CF transmembrane conductance regulator (CFTR) in the appropriate pulmonary cell types. Although CF patients experience multi-organ disease, the chronic bacterial lung infections and associated inflammation are the primary cause of shortened life expectancy. Gene transfer-based therapeutic approaches are feasible, in part, because the airway epithelium is directly accessible by aerosol delivery or instillation. Improvements in standard delivery vectors and the development of novel vectors, as well as emerging technologies and new animal models, are propelling exciting new research forward. Here, we review recent developments that are advancing this field of investigation.

/pdf/genetic-therapies-for-cystic-fibrosis-lung-disease-3i95ln9b1g.pdf

Genetic therapies for cystic fibrosis lung disease

Simple Summary Dogs with cancer often lose their appetite, which leads to weight loss and affects their general health. This study investigated the acceptance of a new therapeutic, nutritionally balanced and calorically dense food by dogs with cancer. The results showed that the dogs readily ate the food, which led to improved caloric intake, increased body weight, and maintained high quality of life. Abstract Diminished appetite and poor eating behavior accompanied by weight loss or cachexia are often reported in dogs living with cancer. This study was conducted to determine the acceptance and eating enthusiasm in dogs with cancer for a new therapeutic, nutritionally balanced, and calorically dense food designed for dogs with cancer. Adult dogs with diagnosis of cancer were recruited from general and oncology practices and were fed the study food for 28 days. Evaluations included physical examination, body weight, food intake, caloric intake, hematology and serum biochemistry, and owner assessments, namely food evaluation, quality of life, and stool scores. The dogs transitioned smoothly and tolerated the food very well. The results showed high food acceptance within the first day, with continued eating enthusiasm over the 28 days. Significant increases in food and caloric intake were observed, with the study food having a positive impact on body weight in dogs that were losing weight and helping to maintain a high quality of life. Blood laboratory parameters remained within reference ranges. Thus, the therapeutic study food was well accepted and efficacious in supporting continued eating and required caloric intake, promoting a healthy weight gain and maintaining a high quality of life in dogs with cancer.

/pdf/acceptance-of-a-novel-highly-palatable-calorically-dense-and-2j86mio5.pdf

Acceptance of a Novel, Highly Palatable, Calorically Dense, and Nutritionally Complete Diet in Dogs with Benign and Malignant Tumors

Introduction Relative supersaturation (RSS) values for urine crystals are a measure of the risk of urinary stone formation and have been shown to be lowered in foods shown to aid in the management of urolithiasis. In order to calculate RSS in pets, computer programs have been developed to calculate RSS and aid in the understanding of stone formation in veterinary medicine. However, some older programs have not been updated for use in animals, and the specific coefficients used are not publically available. One of the first RSS programs was developed in BASIC computer language and published in 1985 which was called EQUIL2. The EQUIL2 program was updated to a compiled version compatible with a PC platform. However, the formulas could not be read or altered. Methods This study evaluates a new program with known coefficients to the original EQUIL2 program. The RSS values of the two programs were compared through a t-test, calculating the r2 from correlation analysis, Lin’s concordance correlation coefficient, and by a Bland–Altman analysis of outputs from the two programs using urine samples from healthy dogs and cats. Results and Discussion Our results show that for both magnesium ammonium phosphate (struvite) and calcium oxalate, the RSS values of the original program could be calculated from the new programs RSS values. Although the actual RSS values were different (as might be expected through the use of the updated coefficients and different thermodynamic stability constants in the calculations) the results were highly correlated, finding elevations and reductions in RSS proportionally in the same urine samples. The current work creates a foundation for using the modernized program to calculate RSS and provides a shared method for understanding the risk of struvite and calcium oxalate stone formation.

/pdf/comparison-of-two-software-programs-used-to-determine-the-16ng258a.pdf

Reshma M. Anthony

Papers

Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

Genetic therapies for cystic fibrosis lung disease

Acceptance of a Novel, Highly Palatable, Calorically Dense, and Nutritionally Complete Diet in Dogs with Benign and Malignant Tumors

Comparison of two software programs used to determine the relative supersaturation of urine ions