Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes.

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.

A traceless linker for aliphatic amines that rapidly and quantitatively fragments after reduction.

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pK a values. Here a traceless self-immolative linker composed of a dithiol-ethyl carbonate connected to a benzyl carbamate (DEC) is presented, which can modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. DEC was able to reversibly modify the lysine residues on CRISPR-Cas9 with either PEG, the cell penetrating peptide Arg10, or donor DNA, and generated Cas9 conjugates with significantly improved biological properties. In particular, Cas9-DEC-PEG was able to diffuse through brain tissue significantly better than unmodified Cas9, making it a more suitable candidate for genome editing in animals. Furthermore, conjugation of Arg10 to Cas9 with DEC was able to generate a self-delivering Cas9 RNP that could edit cells without transfection reagents. Finally, conjugation of donor DNA to Cas9 with DEC increased the homology directed DNA repair (HDR) rate of the Cas9 RNP by 50% in HEK 293T cell line. We anticipate that DEC will have numerous applications in biotechnology, given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Substratum compliance modulates corneal fibroblast to myofibroblast transformation.

PURPOSE: The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor ß1 (TGF-ß1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-ß1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas. METHODS: Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (> 1 gigapascal [GPa]). Cells were cultured in media containing TGF-ß1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot. RESULTS: Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-ß1. CONCLUSIONS: These data indicate that substrate compliance modulates TGF-ß1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.

Substratum topography modulates corneal fibroblast to myofibroblast transformation.

PURPOSE: The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFß-induced myofibroblast transformation of corneal cells. METHODS: Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFß at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed. RESULTS: Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P < 0.002) compared with planar surfaces when exposed to TGFß; the greatest reduction was seen on the 1400 nm surface. Smad7 mRNA expression was significantly greater on all patterned surfaces exposed to TGFß (P < 0.002), whereas cells grown on planar surfaces showed equal or reduced levels of Smad7. Western blot analysis and αSMA immunocytochemical staining demonstrated reduced transition to the myofibroblast phenotype on the 1400 nm surface when compared with cells on a planar surface. CONCLUSIONS: These data demonstrate that nanoscale topographic features modulate TGFß-induced myofibroblast differentiation and αSMA expression, possibly through upregulation of Smad7. It is therefore proposed that in the wound environment, native nanotopographic cues assist in stabilizing the keratocyte/fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.

Phosphatidylinositol-4,5-bisphosphate and phospholipase D-generated phosphatidic acid specify SNARE-mediated vesicle fusion for prospore membrane formation.

The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of proteins is required for eukaryotic intracellular membrane fusions. Vesicle fusion for formation of the prospore membrane (PSM), a membrane compartment that forms de novo during yeast sporulation, requires SNARE function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)], and the activity of the phospholipase D (PLD) Spo14p, which generates phosphatidic acid (PA). The SNARE syntaxin Sso1p is essential for PSM production while the functionally redundant homolog in vegetative growth, Sso2p, is not. We demonstrate that Sso1p and Sso2p bind similarly in vitro to PA or phosphoinositide-containing liposomes and that the conserved SNARE (H3) domain largely mediates PA-binding. Both green fluorescent protein-Sso fusion proteins localize to the developing PSM in wild-type cells and to the spindle pole body in spo14Delta cells induced to sporulate. However, the autoregulatory region of Sso1p binds PI(4,5)P(2)-containing liposomes in vitro with a greater ability than the equivalent region of Sso2p. Overexpression of the phosphatidylinositol-4-phosphate 5-kinase MSS4 in sso1Delta cells induced to sporulate stimulates PSM production; PLD activity is not increased under these conditions, indicating that PI(4,5)P(2) has roles in addition to stimulating PLD in PSM formation. These data suggest that PLD-generated PA and PI(4,5)P(2) collaborate at multiple levels to promote SNARE-mediated fusion for PSM formation.

Phospholipase D function in Saccharomyces cerevisiae.

Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.

Snc1p v-SNARE transport to the prospore membrane during yeast sporulation is dependent on endosomal retrieval pathways.

Vesicular traffic is essential for sporulation in Saccharomyces cerevisiae. The Golgi-associated retrograde protein (GARP) tethering complex is required for retrograde traffic from both the early and late endosomes to the Golgi. Analyses of GARP complex mutants in sporulation reveal defects in meiotic progression and spore formation. In contrast, inactivation of the retromer complex, which mediates vesicle budding and cargo selection from the late endosome, or Snx4p, which is involved in retrieval of proteins from the early endosome, has little effect on sporulation. A retromer GARP double mutant is defective in the formation of the prospore membrane (PSM) that surrounds the haploid nuclei. In the retromer GARP double mutant, PSM precursor vesicles carrying the cargo, Dtr1p, are transported to the spindle pole body (SPB), where PSM formation is initiated. However, the v-SNARE Snc1p is not transported to the SPB in the double mutant, suggesting that the defect in PSM formation is because of the failure to retrieve Snc1p, and perhaps other proteins, from the endosomal pathway. Taken together, these results indicate that retrograde trafficking from the endosome is essential for sporulation by retrieving molecules important for PSM and spore wall formation.