CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and other fungi

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and other fungi

Microneurolysis and decompression of long thoracic nerve injury are effective in reversing scapular winging: Long-term results in 50 cases

<p>Abstract</p> <p>Background</p> <p>Long thoracic nerve injury leading to scapular winging is common, often caused by closed trauma through compression, stretching, traction, direct extrinsic force, penetrating injury, or neuritides such as Parsonage-Turner syndrome. We undertook the largest series of long thoracic nerve decompression and neurolysis yet reported to demonstrate the usefulness of long thoracic nerve decompression.</p> <p>Methods</p> <p>Winging was bilateral in 3 of the 47 patients (26 male, 21 female), yielding a total of 50 procedures. The mean age of the patients was 33.4 years, ranging from 24–57. Causation included heavy weight-lifting (31 patients), repetitive throwing (5 patients), deep massage (2 patients), repetitive overhead movement (1 patient), direct trauma (1 patient), motor bike accident (1 patient), and idiopathic causes (9 patients). Decompression and microneurolysis of the long thoracic nerve were performed in the supraclavicular space. Follow-up (average of 25.7 months) consisted of physical examination and phone conversations. The degree of winging was measured by the operating surgeon (RKN). Patients also answered questions covering 11 quality-of-life facets spanning four domains of the World Health Organization Quality of Life questionnaire.</p> <p>Results</p> <p>Thoracic nerve decompression and neurolysis improved scapular winging in 49 (98%) of the 50 cases, producing "good" or "excellent" results in 46 cases (92%). At least some improvement occurred in 98% of cases that were less than 10 years old. Pain reduction through surgery was good or excellent in 43 (86%) cases. Shoulder instability affected 21 patients preoperatively and persisted in 5 of these patients after surgery, even in the 5 patients with persistent instability who experienced some relief from the winging itself.</p> <p>Conclusion</p> <p>Surgical decompression and neurolysis of the long thoracic nerve significantly improve scapular winging in appropriate patients, for whom these techniques should be considered a primary modality of functional restoration.</p

Challenges and future directions: From panacea to realisation

The pace at which the evolution of technology has occurred within the healthcare context has been unprecedented; however, it has the potential to improve care. The introduction of technological solutions across the care continuum has revolutionised the way care is delivered; however introducing technological solutions and innovations also introduces unintended consequences and new challenges. This chapter emphasises on the challenges for implementing technological solutions in healthcare from an organisational perspective but also on those challenges that emerge following the utilisation of technology (e.g. from a user's perspective) in healthcare. Nevertheless, the technological solutions remain an asset in healthcare, and the development of existing solutions and the introduction of new ones are what we will experience in the future. The challenge is to have health policies in place that facilitate the implementation of technological solutions and making these more person-centric and user friendly without jeopardising ethical principles such as dignity as well as the safety and the privacy of the patient