GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems

Evaluating Supply and Demand Tools and Methodologies

A Ray Marshall Center research team, led by Dr. Christopher King and Dr. Kelly Mikelson, conducted research for the Texas Workforce Investment Council (TWIC) designed to assess currently available workforce supply/demand planning tools in order to help workforce programs and community and technical colleges in Texas to more effectively plan their program offerings.Texas Workforce Investment Council, Texas Workforce CommissionRay Marshall Center for the Study of Human Resource

The United States Energy Policy: As Determined By Non-experts

There are many major environmental issues challenging the world today, including global warming and limited fossil fuel resources. Due to these issues, the methods used by the United States to produce energy and the technology behind these methods are becoming increasingly vital. There are several committees and departments whose role it is to choose what the United States will allow or invest in when it comes to producing and distributing energy to the citizens. These include the Senate Committee on Energy and Natural Resources, the Select Committee on Energy Independence and Global Warming, the House Energy and Commerce Committee, the Federal Energy Regulatory Commission, and the Nuclear Regulatory Commission. These organizations solicit advice from experts in the energy sector. Despite these expert opinions, sometimes energy policies clash with the very objectives they were designed to achieve. This paper seeks to examine the decisions being made in this political arena as well as the impacts of decisions made regarding these policies. The nature of the professional opinions that have been given are also addressed. This paper seeks to identify the value and importance of technological literacy in policy makers as can be achieved by encouraging technical literacy for nonengineering students in the classroom today.ASEE holds the copyright on this document. It may be read by the public free of charge. Authors may archive their work on personal websites or in institutional repositories with the following citation: Foltz, Z., & Kaur, A., & Tushaus, W. H., & Mikelson, C. S., & Skalak, B. V., & Mina, M. (2012, June), "The United States Energy Policy: As Determined By Non-experts." Paper presented at 2012 ASEE Annual Conference & Exposition, San Antonio, Texas. 10.18260/1-2--22106. Copyright 2012 American Society for Engineering Education. Posted with permission

Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.This article is published as Wierson, Wesley A., Jordan M. Welker, Maira P. Almeida, Carla M. Mann, Dennis A. Webster, Melanie E. Torrie, Trevor J. Weiss et al. "Efficient targeted integration directed by short homology in zebrafish and mammalian cells." Elife 9 (2020): e53968. doi: 10.7554/eLife.53968.</p

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems.This is a pre-print made available through bioRxiv, doi: 10.1101/431627.</p

Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems