A non-viral genome editing platform for site-specific insertion of large transgenes

Abstract

Background The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA—despite being successful in E. coli and animal models—still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. Methods Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a ‘gene of interest’. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. Results The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. Conclusion We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

Programmable RNA-Guided Large DNA Transgenesis by CRISPR/Cas9 and Site-Specific Integrase Bxb1

“…Furthermore, while recent CRISPR/Cas9-based approaches, such as Easi (Efficient additions with ssDNA inserts)-CRISPR (using long single-stranded DNA donors) ( Quadros et al, 2017 ), SPRINT (SHERLOCK-based profiling of in vitro transcription)-CRISPR (S-phase pronuclear injection of large DNA) ( Abe et al, 2020 ), and 2C-HR (two-cell homologous recombination)-CRISPR (knock-in of large transgenes in two-cell stage embryos) ( Gu et al, 2018 ), are highly efficient in the range of 1–6 kb, the efficiency of precise introduction of fragments greater than ∼7 kb in length is still not robust. The payload capacity of lentiviral-based techniques is superior (18 kb) ( Chaudhari et al, 2020 ), but these techniques are linked to serious side effects such as genotoxicity ( Montini et al, 2006 ) and immunogenicity ( Nayak and Herzog, 2010 ). Although AAVs have few side effects, their payload capacity is less than 5 kb ( Lai et al, 2010 ).…”

Versatile seamless DNA vector production in E. coli using enhanced phage lambda integrase

“…Distinct advantages of this system are the ease of handling and, due to the employment of enhanced IntC3, the application of various pairs of attachment (att) recombination site derivatives as efficient recombination substrates to yield seamless vectors ranging in size between a few hundred base-pairs to > 10 kb. This flexibility in att site sequences considerably expands the scope of future downstream applications for seamless vectors including site-specific genome editing of higher eukaryotic cells [14,15].…”

STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

“…However, the largest payload reported to be inserted was ~7 kb (Zhu et al, 2014). Alternatively, jC31 and l integrases can introduce larger inserts (up to ~20 kb) but only into pseudo-attP sites already present in the human genome, thus preventing selection of the target site (Chaudhari et al, 2020;Farruggio et al, 2017;Liu et al, 2009). Furthermore, locus-specific silencing of the transgenes occurred in some instances (Farruggio et al, 2017).…”

“…Despite both classes of SSRs being used to genetically modify hPSCs (Chaudhari et al, 2020;Du et al, 2009;Ordovás et al, 2015;Zhu et al, 2014), efficient methods to perform targeted integrations of large DNA payloads (>10 kb) that are also suitable for multiplex assays are still lacking. Cassette exchange strategies, whereby a previously targeted 'landing pad' (LP) cassette containing SSR recognition/attachment sites is used to insert transgenes flanked by corresponding sequences, have been developed for repeated modifications of hPSCs or differentiated progenitor cells (Lv et al, 2018;Pei et al, 2015;Zhu et al, 2014).…”