Multiplex base editing to convert TAG into TAA codons in the human genome

Chen, Yuting; Hysolli, Eriona; Chen, Anlu; S, Casper; Liu, Songlei; Yang, Kevin Yi; Liu, Chenli; Church, George M.

doi:10.1038/s41467-022-31927-8

Cited by 13 publications

(6 citation statements)

References 60 publications

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“…In Box 1, we conclude our observations for future genome designs. Follow-up works might translate and validate our results in ongoing prokaryotic and eukaryotic genome recoding projects 18,19,21,26,29,42,[74][75][76] .…”

Section: Discussionsupporting

confidence: 63%

Synthetic genomes unveil the effects of synonymous recoding

Nyerges,

Chiappino-Pepe,

Budnik

et al. 2024

Preprint

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Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1–4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement—recoding—is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a syntheticEscherichia coligenome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organism’s genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

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Section: Discussionsupporting

confidence: 63%

Synthetic genomes unveil the effects of synonymous recoding

Nyerges,

Chiappino-Pepe,

Budnik

et al. 2024

Preprint

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“…Additionally, introduction of multiple gene edits in one cell line will need to balance effectiveness with avoidance of off-target effects and other adverse events. 53 , 54 …”

Section: Resultsmentioning

confidence: 99%

Diminished Immune Cell Adhesion in Hypoimmune ICAM-1 Knockout Pluripotent Stem Cells

Saha,

Haynes,

Del Rio

et al. 2024

Preprint

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Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g., T cell) immune responses, but have largely not addressed the innate immune cells (e.g., monocytes, neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies, we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responsesin vitroand in longerin vivoretention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding, thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases.HighlightsAntibody blocking of ICAM-1 on human pluripotent stem cell-derived cells inhibits immune cell adhesionCRISPR/Cas9 knock-out of ICAM-1 ablates surface and secreted ICAM-1 protein and inhibits immune adhesionICAM-1 knock-out results in decreased T cell proliferative responses to human pluripotent stem cell-derived graftsin vitro, and resistance to immune-mediated graft lossin vivoAddition of ICAM-1 knock-out to first generation MHC knock-out human pluripotent stem cells confers protection against immune adhesionGraphical AbstractICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse.Abbreviations: PSC-EC – pluripotent stem cell-derived endothelial cells; KO – knock-out; dSMAC – distal supramolecular activation complex; pSMAC – peripheral supramolecular activation complex; cSMAC – central supramolecular activation complex.

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“…showed base editing (TAG to TAA) of 33 target sites (out of the 47) via a single transfection. 85 Several other studies improved base editing by narrowing the editing window, enhancing DNA specificity, and developing different PAM compatibilities, using a different variant of APOBEC1 and small molecule dependence. 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 …”

Section: Emerging Precise Gene Correction Technologiesmentioning

confidence: 99%