Piezoelectric excitation and acoustic detection of thin film polymer membrane vibrations.

A simple method of measuring the vibrational response of a thin film membrane was developed. Piezoelectric excitation and acoustic detection (using a microphone) allowed the vibrational spectra of thin membranes to be measured in the kHz range. Vibrational frequencies were used to determine Young's modulus in thin (µm) solvent tensioned films of polydimethylsiloxane and to measure tension in ultrathin polystyrene films. Simulations of membrane motion generated vibrational spectra that agreed with the results of experiments for different membrane shapes.

Thickness dependence of telephone cord blister formation in solvent swollen films of polydimethylsiloxane.

The thickness dependence of telephone cord blister formation in thin films of solvent swollen polydimethylsiloxane (PDMS) was studied using a simple imaging setup. Chloroform was deposited on top of PDMS that had been spin coated on to glass slides coated with a thin wax layer. After an initial thickness dependent nucleation time, straight-sided blisters were observed to form on the films. These later developed into sinusoidal telephone cord blisters. Movies of the growing telephone cord blisters were recorded at 200 fps for PDMS films with thickness values in the range 38 < h ≤ 223 µm. Software written in Python was used to analyse the movies and to extract the thickness dependence of the width of the telephone cord blisters as well as the wavelength of the sinusoidal corrugations and the blister growth rates. Data were interpreted in the context of theories of buckling and dynamic fracture mechanics.

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants.

BACKGROUND: Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. RESULTS: To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. CONCLUSIONS: Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

Imaging biopsy composition at ACL reconstruction.

PURPOSE: Early-stage osteoarthritis (OA) includes glycosaminoglycan (GAG) loss and collagen disruption that cannot be seen on morphological magnetic resonance imaging (MRI). T1ρ MRI is a measurement that probes the low-frequency rate of exchange between protons of free water and those from water associated with macromolecules in the cartilage's extracellular matrix. While it has been hypothesized that increased water mobility resulting from early osteoarthritic changes cause elevated T1ρ MRI values, there remain several unknown mechanisms influencing T1ρ measurements in cartilage. The purpose of this work was to relate histological and biochemical metrics directly measured from osteochondral biopsies and fluid specimens with quantitative MRI-detected changes of in vivo cartilage composition. PATIENTS AND METHODS: Six young patients were enrolled an average of 41 days after acute anterior cruciate ligament (ACL) rupture. Femoral trochlear groove osteochondral biopsies, serum, and synovial fluid were harvested during ACL reconstruction to complement a presurgery quantitative MRI study (T1ρ, T2, delayed gadolinium-enhanced MRI of cartilage [dGEMRIC] relaxation times). A high-resolution MRI scan of the excised osteochondral biopsy was also collected. Analyses of in vivo T1ρ images were compared with ex vivo T1ρ imaging, GAG assays and histological GAG distribution in the osteochondral biopsies, and direct measures of bone and cartilage turnover markers and "OA marker" 3B3 in serum and synovial fluid samples. CONCLUSION: T1ρ relaxation times in patients with a torn ACL were elevated from normal, indicating changes consistent with general fluid effusion after blunt joint trauma. Increased chondrogenic progenitor cell (CPC) production of chondroprotective lubricin may relate to cartilage surface disruption by blunt trauma and CPC amplification of joint inflammation. Disparity between ex vivo and matched in vivo MRI of trochlear cartilage suggests MRI signal differences that may be related to the synovial fluid environment. T1ρ is emerging as a promising MRI biomarker to relate noninvasive measures of whole-joint condition and cartilage composition to direct measures of cartilage changes in the acute phase of joint injuries.