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      Genome-wide inactivation of porcine endogenous retroviruses (PERVs).

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          Abstract

          The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.

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          Optimization of scarless human stem cell genome editing

          Luhan Yang, Marc Guell, Susan Byrne … (2013)
          Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.
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            PiggyBac transposon-mediated gene transfer in human cells.

            Matthew Wilson, Craig J Coates, Alfred George (2007)
            Transposons are mobile genetic elements that can be used to integrate transgenes into host cell genomes. The piggyBac transposon system has been used for transgenesis of insects and for germline mutagenesis in mice. We compared transposition activity of piggyBac with Sleeping Beauty (SB), a widely used transposon system for preclinical gene therapy studies. An engineered piggyBac transposon with minimal length 5' and 3' terminal repeats exhibited greater transposition activity in transfected cultured human cells than a well-characterized hyperactive SB system. PiggyBac excision was very precise as evidenced by the typical absence of "footprint" mutations at the site of transposon excision. We mapped 575 piggyBac integration sites in human cells to determine site selectivity of genomic integration. PiggyBac demonstrated non-random integration site selectivity that differed from that previously reported for SB, including a higher preference for integrations in regions surrounding transcriptional start sites and within long terminal repeat elements. Importantly, overproduction inhibition was not observed with piggyBac, a major limitation of the SB system. This permitted the generation of combination "helper-independent" piggyBac transposase-transposon vectors that exhibited a 2-fold increase of transposition activity in human cells as compared with cells transfected with separate transposon and transposase plasmids. We conclude that piggyBac is a transposon system with certain properties, including high efficiency and lack of overproduction inhibition that are advantageous in preclinical development of transposon-based gene therapy.
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              Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA).

              Marc Guell, Luhan Yang, George Church (2014)
              Clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies have revolutionized human genome engineering and opened countless possibilities to basic science, synthetic biology and gene therapy. Albeit the enormous potential of these tools, their performance is far from perfect. It is essential to perform a posterior careful analysis of the gene editing experiment. However, there are no computational tools for genome editing assessment yet, and current experimental tools lack sensitivity and flexibility. We present a platform to assess the quality of a genome editing experiment only with three mouse clicks. The method evaluates next-generation data to quantify and characterize insertions, deletions and homologous recombination. CRISPR Genome Analyzer provides a report for the locus selected, which includes a quantification of the edited site and the analysis of the different alterations detected. The platform maps the reads, estimates and locates insertions and deletions, computes the allele replacement efficiency and provides a report integrating all the information. CRISPR-GA Web is available at http://crispr-ga.net. Documentation on CRISPR-GA instructions can be found at http://crispr-ga.net/documentation.html mguell@genetics.med.harvard.edu. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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                Author and article information

                Journal
                Journal ID (iso-abbrev): Science
                Title: Science (New York, N.Y.)
                ISSN (Electronic): 1095-9203
                ISSN (Print): 0036-8075
                Publication date (Electronic): Nov 27 2015
                Volume: 350
                Issue: 6264
                Affiliations
                [1 ] Department of Genetics, Harvard Medical School, Boston, MA, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA. eGenesis Biosciences, Boston, MA 02115, USA. gchurch@genetics.med.harvard.edu luhan.yang@egenesisbio.com.
                [2 ] Department of Genetics, Harvard Medical School, Boston, MA, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA. eGenesis Biosciences, Boston, MA 02115, USA.
                [3 ] Department of Genetics, Harvard Medical School, Boston, MA, USA. College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
                [4 ] Department of Genetics, Harvard Medical School, Boston, MA, USA.
                [5 ] Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
                [6 ] Transplant Infectious Disease and Compromised Host Program, Massachusetts General Hospital, Boston, MA 02115, USA.
                Article
                Publisher Item ID: science.aad1191
                DOI: 10.1126/science.aad1191
                PubMed ID: 26456528
                SO-VID: 8a4a5c3d-9251-4c26-9a89-6e05c0c7c61b
                Copyright © Copyright © 2015, American Association for the Advancement of Science.
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