~150 words and referenced]Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies 1-4 . We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells 5 . Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to 'shuttle' the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for nonviral genome reprogramming that we successfully applied in regulatory T cells (Tregs), -T cells, B cells, NK cells, and primary and iPS-derived 6 hematopoietic stem progenitor cells (HSPCs).We recently reported an approach to reprogram human T cells with CRISPR-based genome targeting without the need for viral vectors 5 . However, many research and clinical applications still depend upon improved efficiency, cell viability, and generalizability of non-viral genome targeting across cell types 1-4 . We previously found that varying the relative concentrations of both Cas9 RNP and HDR template had significant effects on targeting efficiency
Although DSBs are known to initiate transcriptional changes, less is understood 56 about the role of translation in the DNA damage response. A purely transcriptional 57 4 reaction to a genetic insult leaves a gap in response, potentially exposing a cell to the 58 impact of damaged DNA during a critical time window in which damage had raised an 59 alarm but newly transcribed mRNAs have not accumulated. While transcriptional 60 changes can modulate protein abundance hours or days after a genomic insult, 61 translational control can enact regulatory programs within minutes of an environmental 62 stress (Andreev et al., 2015;Sidrauski et al., 2015). 63We thus sought to characterize how cells respond to DNA damage at the 64 translation level, and in particular, how cells respond to a single double-strand break 65 during Cas9-mediated genome editing. We serendipitously found that cells temporarily 66 deplete core ribosomal proteins, RPS27A and RPL40, in response to dsDNA damage. 67 RPS27A and RPL40 are regulated post-transcriptionally and in a p53-independent 68 manner, and their depletion persists days after the initial genomic lesion with Cas9. We 69 also found that both non-specific double-strand breaks as well as single, targeted 70 double-strand breaks reduce translation via eukaryotic initiation factor 2 alpha (eIF2α) 71 phosphorylation, and that modulating the downstream effects of eIF2α phosphorylation 72 during Cas9 editing leads to different repair outcomes. Ribosome profiling and RNA-seq 73 data from Cas9-edited cells suggest that cells mount a translation response to dsDNA 74 damage that precedes transcriptional changes. Our data demonstrate that Cas9-75 mediated genome editing can trigger temporary ribosome remodeling and translational 76 shutdown in response to DNA double-strand breaks. 77 5 Results 78Ribosome proteins RPS27A and RPL40 are downregulated after genome editing 79 with Cas9 80 While investigating changes in ubiquitin gene expression after DNA damage, we 81 serendipitously observed that the two ribosomal proteins encoded as fusion proteins 82 with ubiquitin, RPS27A (eS31) and RPL40 (eL40), are downregulated after Cas9-guide 83 RNA (gRNA) ribonucleoprotein (RNP) nucleofection ( Figure 1A). This downregulation 84 was apparent as late as 48-72 hours after nucleofection, even though at this point Cas9 85 was largely absent from the cell (Figure 1B) and genomic formation of indels was 86 completed (Figure 1C). We found that RPS27A levels recovered 96 hours after 87 nucleofection and RPL40 levels were beginning to increase within 72 hours ( Figure 88 1A), suggesting that the cell resets protein expression three to four days after editing 89 ( Figure S1A). 90 Downregulation of RPS27A and RPL40 depended on the DNA double-strand 91 break, as catalytically inactive dCas9 did not provoke a similar response (Figure 1A). 92The guide RNA used in this experiment targeted a non-coding region of the JAK2 gene 93 (sgIntron), and JAK2 levels remain unchanged after Cas9 nucleofection (Figure S1B). 94Our data the...
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