Detection of CRISPR-mediated genome modifications through altered methylation patterns of CpG islands.

BACKGROUND: The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision. RESULTS: In this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level. CONCLUSIONS: With advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence.

Optical properties of superconducting nanowire single-photon detectors.

We measured the optical absorptance of superconducting nanowire single photon detectors. We found that 200-nm-pitch, 50%-fill-factor devices had an average absorptance of 21% for normally-incident front-illumination of 1.55-microm-wavelength light polarized parallel to the nanowires, and only 10% for perpendicularly-polarized light. We also measured devices with lower fill-factors and narrower wires that were five times more sensitive to parallel-polarized photons than perpendicular-polarized photons. We developed a numerical model that predicts the absorptance of our structures. We also used our measurements, coupled with measurements of device detection efficiencies, to determine the probability of photon detection after an absorption event. We found that, remarkably, absorbed parallel-polarized photons were more likely to result in detection events than perpendicular-polarized photons, and we present a hypothesis that qualitatively explains this result. Finally, we also determined the enhancement of device detection efficiency and absorptance due to the inclusion of an integrated optical cavity over a range of wavelengths (700-1700 nm) on a number of devices, and found good agreement with our numerical model.

Direct index of refraction measurements at extreme-ultraviolet and soft-x-ray wavelengths.

Coherent radiation from undulator beamlines has been used to directly measure the real and imaginary parts of the index of refraction of several materials at both extreme-ultraviolet and soft-x-ray wavelengths. Using the XOR interferometer, we measure the refractive indices of silicon and ruthenium, essential materials for extreme-ultraviolet lithography. Both materials are tested at wavelength (13.4 nm) and across silicon's L2 (99.8 eV) and L3 (99.2 eV) absorption edges. We further extend this direct phase measurement method into the soft-x-ray region, where measurements of chromium and vanadium are performed around their L3 absorption edges at 574.1 and 512.1 eV, respectively. These are the first direct measurements, to our knowledge, of the real part of the index of refraction made in the soft-x-ray region.

Nanowire single-photon detector with an integrated optical cavity and anti-reflection coating.

We have fabricated and tested superconducting single-photon detectors and demonstrated detection efficiencies of 57% at 1550-nm wavelength and 67% at 1064 nm. In addition to the peak detection efficiency, a median detection efficiency of 47.7% was measured over 132 devices at 1550 nm. These measurements were made at 1.8K, with each device biased to 97.5% of its critical current. The high detection efficiencies resulted from the addition of an optical cavity and anti-reflection coating to a nanowire photodetector, creating an integrated nanoelectrophotonic device with enhanced performance relative to the original device. Here, the testing apparatus and the fabrication process are presented. The detection efficiency of devices before and after the addition of optical elements is also reported.

Diffractive optical elements based on Fourier optical techniques: a new class of optics for extreme ultraviolet and soft x-ray wavelengths.

A diffractive optical element, based on Fourier optics techniques, for use in extreme ultraviolet/soft x-ray experiments has been fabricated and demonstrated. This diffractive optical element, when illuminated by a uniform plane wave, will produce two symmetric off-axis first-order foci suitable for interferometric experiments. The efficiency of this optical element and its use in direct interferometric determination of optical constants are also discussed. Its use in direct interferometric determination of optical constants is also referenced. Its use opens a new era in the use of sophisticated optical techniques at short wavelengths.