RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

/pdf/highly-efficient-rna-guided-genome-editing-in-human-cells-1dwagla9ju.pdf

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

/pdf/analysis-of-off-target-effects-of-crispr-cas-derived-rna-4qj3ty11fb.pdf

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Visible-light photocatalysis has evolved over the last decade into a widely used method in organic synthesis. Photocatalytic variants have been reported for many important transformations, such as cross-coupling reactions, α-amino functionalizations, cycloadditions, ATRA reactions, or fluorinations. To help chemists select photocatalytic methods for their synthesis, we compare in this Review classical and photocatalytic procedures for selected classes of reactions and highlight their advantages and limitations. In many cases, the photocatalytic reactions proceed under milder reaction conditions, typically at room temperature, and stoichiometric reagents are replaced by simple oxidants or reductants, such as air, oxygen, or amines. Does visible-light photocatalysis make a difference in organic synthesis? The prospect of shuttling electrons back and forth to substrates and intermediates or to selectively transfer energy through a visible-light-absorbing photocatalyst holds the promise to improve current procedures in radical chemistry and to open up new avenues by accessing reactive species hitherto unknown, especially by merging photocatalysis with organo- or metal catalysis.

Visible-Light Photocatalysis: Does It Make a Difference in Organic Synthesis?

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

/pdf/a-guide-to-super-resolution-fluorescence-microscopy-28ekkdaf4n.pdf

A guide to super-resolution fluorescence microscopy

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.

/pdf/evaluation-of-fluorophores-for-optimal-performance-in-2kfvou9h34.pdf

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Besides being versatile and fast, rapidSTORM is easy to use, deploy, inspect and extend. It is open source and based on widespread, mature, portable and open technologies such as C++, the GNU tool chain and wxWidgets. A graphical user interface and user manual allow quick acquaintance. Automated prerelease validation and wide use on a range of biological targets ensure reliable results. rapidSTORM is regularly updated, and both source code and compiled packages are available from our website at http://www.superresolution.biozentrum.uni-wuerzburg.de/.

rapidSTORM: accurate, fast open-source software for localization microscopy.

Live-cell dSTORM with SNAP-tag fusion proteins.

In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with approximately 20 nm optical resolution can be completed in less than 10 s.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2818.2009.03287.x

Steve Wolter

Papers

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

rapidSTORM: accurate, fast open-source software for localization microscopy.

Live-cell dSTORM with SNAP-tag fusion proteins.

Real-time computation of subdiffraction-resolution fluorescence images.

The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging