Member (Student), Cardiovascular Institute
Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus.
Nature biomedical engineering
The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.
View details for DOI 10.1038/s41551-023-01037-x
View details for PubMedID 37127707
Advances in CRISPR therapeutics.
Nature reviews. Nephrology
The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR-Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.
View details for DOI 10.1038/s41581-022-00636-2
View details for PubMedID 36280707
Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.
2022; 13 (1): 2766
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.
View details for DOI 10.1038/s41467-022-30546-7
View details for PubMedID 35589813
The chromatin organization of a chlorarachniophyte nucleomorph genome.
2022; 23 (1): 65
BACKGROUND: Nucleomorphs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features.RESULTS: In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organization for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleomorph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions and show the relative contact frequencies between the multiple genomic compartments of distinct origin that B. natans cells contain.CONCLUSIONS: We provide the first study of a nucleomorph genome using modern functional genomic tools, and derive numerous novel insights into the physical and functional organization of these unique genomes.
View details for DOI 10.1186/s13059-022-02639-5
View details for PubMedID 35232465