- Email: [email protected]
136. Nuclease-Assisted Vector Integration (NAVI): A Robust Platform for Multiplexed Targeted Genome Engineering
Targeted Genome Editing I characterize the genetic alterations. To address drug response and resistance in sarcoma, we have established a genome-wide insertional mutagenesis screening approach. To this end, a panel of STS cell lines is screened for the sensitivity towards drugs currently used for standard therapy of STS. Sensitive cell lines will be treated with transforming lentiviral vectors to identify genes involved in drug resistance. The genetic alterations potentially involved in acquired resistance mechanisms will be functionally characterized using isogenic cell lines created by gene editing approaches. Towards this end, we have successfully employed transcription activator-like effector nucleases and CRISPR/Cas9 system RNA guided endonucleases targeting the BRAF locus to introduce the previously described V600E mutation in 293T cells, as a “proof of concept”. Analysis of the target site showed that 31 % of the sequences were positive for the mutation. The mutation was also confirmed in single cell clones by tetra-primer ARMS-PCR and western blot using an antibody specific for V600E mutated B-Raf. To test the method in a model relevant to STS, we applied gene editing to introduce a previously undescribed KRT8 mutation found in STS patients into a sarcoma cell line. For this mutation, wild type and D10A-mutated Cas9 expressing constructs were generated to allow use of single nucleases and paired nickases. We applied single stranded oligodeoxynucleotides as donor template for KRT8. Using high-throughput sequencing, we detected successful recombination of the mutated KRT8 gene locus in 0.6 % of the treated STS cells. We are using improved guide RNA and donor design as well as compounds affecting non-homologous end-joining and homologous recombination to improve efficiency of successful recombination. To summarize, we successfully edited wild-type BRAF to BRAF V600E in 293T cells and showed that gene editing of KRT8 is possible in sarcoma cells. The methodology will be further optimized to characterize genes involved in resistance to therapy identified in the genome-wide screens. In addition, our gene editing approaches will also be used to model genetic alterations identified in STS patients by next generation sequencing. Our findings will provide interesting insights in nominating drugs for combinatorial therapy to circumvent drug resistance in STS.
135. Optimization of Dual-gRNA Lentiviral Vectors for Targeted Genomic Deletions
Giulia Pavani1, Annalisa Lattanzi1, Fatima Amor1, Chiara Antoniani2, Fulvio Mavilio1, Annarita Miccio2, Mario Amendola1 1 Genome Editing, Genethon, Evry, France, 2Laboratory of Chromatin and Gene Regulation During Development, Imagine Institute, Paris, France CRISPR-Cas9 technology is a powerful tool for genome editing based therapeutic approaches. Despite its potential, the delivery of Cas9 components into primary cells, such as hematopoietic stem progenitor cells (HSPC), is still a major challenge. In addition, several applications - e.g. the use of Cas9-nickase and the generation of genomic deletions/inversions - require the delivery and expression of two different gRNA. Lentiviral vectors (LV) can efficiently transduce HSPC, but the presence and the orientation of direct repeated elements in the gRNA expression cassettes can potentially trigger recombination events that affect vector stability. To address this issue, we designed different LV encoding for two gRNA pairs, which generate deletions of different size (3 and 13 kb) in the betaglobin gene cluster. gRNA expression was driven by murine and human U6 promoters, having little sequence similarity, to avoid potential recombination events of the LV genome. To further reduce LV rearrangements and optimize expression levels, gRNA cassettes were positioned in different orientations with respect to each other (LV Inward, Tandem and Outward). We compared LV from different vector batches in HCT116 cells and we observed that Tandem configuration resulted in higher viral titers and infectivity, although this difference was not statistically significant. Analysis of proviral Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
integrity on genomic DNA of transduced cells showed the intact dual-gRNA cassette and no sign of recombination for all the LV configurations. To understand if different orientations could affect gRNA expression (measured by RT-qPCR) and consequently deletion/ inversion efficiency, we co-transduced erythroleukemic K562 cells with LV-Cas9-Blast and the three different LV. Surprisingly, LV Inward showed poor gRNA expression, negligible deletion and inversion frequency and low efficiency of InDel formation at each gRNA target site (2.4 % and 6.8%). Conversely, LV Tandem and Outward configurations allowed efficient cell transduction and significant and comparable levels of gRNA expression. As a results, we observed a good deletion frequency (11.0% ±1.8 of total alleles for LV Tandem and 12.3 %± 2.5 for LV Outward), a lower proportion of inversions events (5.4% ±1.3 for LV Tandem and 3.8 %± 0.4 for LV Outward) and up to 58% of InDels events at each gRNA target sites. Comparable results were also obtained in adult HSPC-derived erythroid cell line (HUDEP-2). Overall, our study indicates LV Tandem and LV Outward as promising tools for genome editing of primary cells; both vectors are now being evaluated in primary HSPC.
136. Nuclease-Assisted Vector Integration (NAVI): A Robust Platform for Multiplexed Targeted Genome Engineering Alexander Brown, Wendy S. Woods, Pablo Perez-Pinera Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL
The expansion of genome engineering spurred by CRISPR/Cas9 continues to accelerate. While mutagenesis generated via NHEJ remains a highly efficient and effective strategy for select applications, the insertion of large or complex sequences and the ability to easily select for modified cells often necessitates the use of homology directed repair (HDR) based strategies. The time-consuming construction of donor vectors for HDR gene editing is often technically challenging, costly, and leads to poor modification rates. By using customized single-stranded oligonucleotides (ssODN) the efficiency of gene editing increases, but the scale of possible genetic changes is greatly diminished. Additionally, as both donor vectors and ssODN require two discontiguous regions of homology, neither is well suited to multiplexing. Here, we present Nuclease-Assisted Vector Integration (NAVI) as a unique strategy to bypass HDR and the need for customized donor vectors required for traditional genome editing technologies. We demonstrate, through multiplexed insertion of a single plasmid into multiple loci, that NAVI eliminates the need for homologous sequence within donor vectors. Furthermore, by employing a single and universal guide-RNA, multiple vectors harboring different selection markers were integrated into distinct loci simultaneously, greatly facilitating production of isogenic mammalian cell lines. We were able to integrate sequences ranging from 3-50 kbp, but no upper limit was identified. Through the elimination of the HDR bottleneck and increased efficiency of editing, generation of double knockouts within two loci using NAVI takes approximately 3 weeks from design to completion, at minimal cost. Additionally, as NAVI is independent of homology, vector integration at double strand breaks occurs in either the plus or minus orientation. Finally, the resulting sequence of the juncture of genomic DNA with the vector is variable. We conclude that NAVI, despite sacrificing single base pair resolution, can be readily adapted for use in a variety of research and therapeutic platforms, due to its greatly enhanced versatility, ease of use, efficiency, and robust multiplexing capabilities for targeted integration of large sequences within mammalian genomes.
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135. Optimization of Dual-gRNA Lentiviral Vectors for Targeted Genomic Deletions
Giulia Pavani1, Annalisa Lattanzi1, Fatima Amor1, Chiara Antoniani2, Fulvio Mavilio1, Annarita Miccio2, Mario Amendola1 1 Genome Editing, Genethon, Evry, France, 2Laboratory of Chromatin and Gene Regulation During Development, Imagine Institute, Paris, France CRISPR-Cas9 technology is a powerful tool for genome editing based therapeutic approaches. Despite its potential, the delivery of Cas9 components into primary cells, such as hematopoietic stem progenitor cells (HSPC), is still a major challenge. In addition, several applications - e.g. the use of Cas9-nickase and the generation of genomic deletions/inversions - require the delivery and expression of two different gRNA. Lentiviral vectors (LV) can efficiently transduce HSPC, but the presence and the orientation of direct repeated elements in the gRNA expression cassettes can potentially trigger recombination events that affect vector stability. To address this issue, we designed different LV encoding for two gRNA pairs, which generate deletions of different size (3 and 13 kb) in the betaglobin gene cluster. gRNA expression was driven by murine and human U6 promoters, having little sequence similarity, to avoid potential recombination events of the LV genome. To further reduce LV rearrangements and optimize expression levels, gRNA cassettes were positioned in different orientations with respect to each other (LV Inward, Tandem and Outward). We compared LV from different vector batches in HCT116 cells and we observed that Tandem configuration resulted in higher viral titers and infectivity, although this difference was not statistically significant. Analysis of proviral Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
integrity on genomic DNA of transduced cells showed the intact dual-gRNA cassette and no sign of recombination for all the LV configurations. To understand if different orientations could affect gRNA expression (measured by RT-qPCR) and consequently deletion/ inversion efficiency, we co-transduced erythroleukemic K562 cells with LV-Cas9-Blast and the three different LV. Surprisingly, LV Inward showed poor gRNA expression, negligible deletion and inversion frequency and low efficiency of InDel formation at each gRNA target site (2.4 % and 6.8%). Conversely, LV Tandem and Outward configurations allowed efficient cell transduction and significant and comparable levels of gRNA expression. As a results, we observed a good deletion frequency (11.0% ±1.8 of total alleles for LV Tandem and 12.3 %± 2.5 for LV Outward), a lower proportion of inversions events (5.4% ±1.3 for LV Tandem and 3.8 %± 0.4 for LV Outward) and up to 58% of InDels events at each gRNA target sites. Comparable results were also obtained in adult HSPC-derived erythroid cell line (HUDEP-2). Overall, our study indicates LV Tandem and LV Outward as promising tools for genome editing of primary cells; both vectors are now being evaluated in primary HSPC.
136. Nuclease-Assisted Vector Integration (NAVI): A Robust Platform for Multiplexed Targeted Genome Engineering Alexander Brown, Wendy S. Woods, Pablo Perez-Pinera Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL
The expansion of genome engineering spurred by CRISPR/Cas9 continues to accelerate. While mutagenesis generated via NHEJ remains a highly efficient and effective strategy for select applications, the insertion of large or complex sequences and the ability to easily select for modified cells often necessitates the use of homology directed repair (HDR) based strategies. The time-consuming construction of donor vectors for HDR gene editing is often technically challenging, costly, and leads to poor modification rates. By using customized single-stranded oligonucleotides (ssODN) the efficiency of gene editing increases, but the scale of possible genetic changes is greatly diminished. Additionally, as both donor vectors and ssODN require two discontiguous regions of homology, neither is well suited to multiplexing. Here, we present Nuclease-Assisted Vector Integration (NAVI) as a unique strategy to bypass HDR and the need for customized donor vectors required for traditional genome editing technologies. We demonstrate, through multiplexed insertion of a single plasmid into multiple loci, that NAVI eliminates the need for homologous sequence within donor vectors. Furthermore, by employing a single and universal guide-RNA, multiple vectors harboring different selection markers were integrated into distinct loci simultaneously, greatly facilitating production of isogenic mammalian cell lines. We were able to integrate sequences ranging from 3-50 kbp, but no upper limit was identified. Through the elimination of the HDR bottleneck and increased efficiency of editing, generation of double knockouts within two loci using NAVI takes approximately 3 weeks from design to completion, at minimal cost. Additionally, as NAVI is independent of homology, vector integration at double strand breaks occurs in either the plus or minus orientation. Finally, the resulting sequence of the juncture of genomic DNA with the vector is variable. We conclude that NAVI, despite sacrificing single base pair resolution, can be readily adapted for use in a variety of research and therapeutic platforms, due to its greatly enhanced versatility, ease of use, efficiency, and robust multiplexing capabilities for targeted integration of large sequences within mammalian genomes.
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