Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites

Advances in Engineering the Fly Genome with the CRISPR-Cas System.

Drosophila has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in Drosophila and highlight key areas for future advances

PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.

Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets

Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

The domestication of the probiotic bacterium Lactobacillus acidophilus

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair.

Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording

SMAJ-tautia aiheuttavan mutaation korjaus potilaan lihaksen kantasoluissa CRISPR-Cas9 geenieditointitekniikalla

Vuonna 2011 Suomesta löydettiin uusi perinnöllinen sairaus, jonka todettiin olevan lähtöisin Pohjois-Karjalan alueelta ja siten osa suomalaista tautiperimää. Taudin nimi on Jokela tyypin spinaalinen lihasatrofia (Spinal Muscular Atrophy of Jokela Type = SMAJ) ja sitä sairastaa yhteensä arviolta 200–400 potilasta. SMAJ on autosomaalisesti vallitseva, alempien liikehermosolujen rappeumasairaus, jonka aiheuttaa pistemutaatio c.197G>T geenissä CHCHD10 ja yhden aminohapon vaihtuminen p.G66V vastaavassa proteiinissa. CHCHD10 on saman nimisestä geenistä tuotettu proteiini, jota esiintyy mitokondrioiden ulko- ja sisäkalvon välissä. Sen tarkkaa toimintaa ei tunneta, eikä miten mutaatio siihen vaikuttaa. Taudin aiheuttavan mekanismin selvittäminen on kuitenkin olennaisen tärkeää, jotta mahdollisia hoitoja ja diagnostisia testejä on mahdollista kehittää. Tutkielman tavoite on korjata tämä mutaatio heterotsygootin SMAJ-potilaan myoblasteissa (lihassolun esiaste) käyttämällä CRISPR-Cas9 geenieditointi teknologiaa. Korjaamalla mutaatio, voidaan luoda solulinja, joka on muuten täysin identtinen potilaan solujen kanssa, mutta mutaatio geenissä CHCHD10 on korjattu vastaamaan normaalia. Vertailemalla tätä solulinjaa potilaan soluihin, voidaan selvittää ainoastaan mutaatiosta johtuvat erot soluissa. Tutkielmassa pyritään selvittämään taudin aiheuttavan mutaation vaikutuksia ihmisen lihassoluissa. Myoblasti-solujen CRISPR-Cas9 geenieditointi ei ole yleistä, sillä usein vastaavan kaltaisia tutkimuksia tehdään indusoiduilla kantasoluilla, joista kohdekudoksen soluja on helppo erilaistaa. Nyt käytettävissä oli kuitenkin potilaan tutkimuskäyttöön luovuttamia lihaksen kantasoluja ja oli mielenkiintoista tutkia mitokondriaalisen CHCHD10 proteiinin aiheuttamaa tautia mitokondriorikkaissa lihassoluissa. CRISPR-Cas9 ribonukleproteiinikompleksia (RNP) ja sitä vastaavaa korjaustemplaattia käytettiin mutaation korjaamisessa. RNP-kompleksi transfektoitiin soluihin elektroporaation avulla, jota ennen elektroporaatio-olosuhteet optimoitiin lihassoluille edullisiksi. Elektroporoitujen solulinjojen geenieditoinnin onnistuminen arvioitiin sekä restriktioentsyymianalyysin, että Synthego ICE CRISPR internettyökalun avulla. Klonaaliset solulinjat luotiin fluoresenssiavusteisella solun lajittelu teknologialla (FACS) ja manuaalisesti poimimalla kolonioita solu maljoilta. Kloonien genotyypit selvitettiin Sanger sekvensoimalla ja arvioitiin off-target geenieditoinnin varalta. Yksi korjattu solulinja saatiin valmistettua ja geenieditointiprosessin optimisaatio myoblasti-soluille onnistui. Tuotetun isogeenisen solulinjan avulla voidaan tulevaisuudessa tutkia CHCHD10:n proteiini- ja mRNA-tasojen eroja verrattuna potilassolulinjaan, ja näin saada arvokasta informaatiota sairauden vaikutuksista lihassoluissa. Tulevaisuuden tehtäviin lukeutuu myös mutaation vaikutuksien hermolihasliitokseen tutkiminen indusoiduista kantasoluista erikoistettujen hermosolujen avulla. Yhteissolukulttuurit mahdollistavat editoitujen myoblastien, sekä hermosolujen kasvattamisen ja hermolihasliitoksen tutkimisen in vitro. Hermolihasliitos on tärkeä yhdistäjä alempien liikehermosolujen ja luustolihasten välissä ja mutaation vaikutuksen tutkiminen voisi valaista SMAJ-tautimekanismia entisestään.Spinal muscular atrophy of Jokela type (SMAJ) is an autosomal dominant motor-neuron disease caused by a missense mutation c.197G>T, p.G66V in the gene CHCHD10. Coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is a nuclear-encoded mitochondrial protein located in the intermembrane space (IMS) of mitochondria with an unknown exact function and disease-causing mechanism. In this project, the overarching aim was to correct a heterozygous SMAJ-causing mutation in patient myoblast cells with CRISPR-Cas9 genome editing. The goal was to create a genetically identical, isogenic, cell line to study only the effects of the mutation on cellular phenotype in vitro. Human myoblast cells isolated from patient biopsies provide the most pertinent experimental model to study neuromuscular atrophy-associated mutations in their natural genomic environment. More specific aims included genome editing optimization with myoblast cells, since it is not as widely conducted as with some other cell types, such as iPSCs. CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology-directed repair (HDR) in the genome of patient-derived myoblast cells and correct the mutation. After optimization of electroporation conditions for myoblast cells, guide RNAs were designed and transfected into patient myoblasts. Clonal cell lines were made by utilizing techniques such as fluorescence adjusted cell sorting (FACS) and manual colony picking. The success and precision of genome editing were analyzed by Sanger sequencing, comparing the performance of the different guide RNAs with restriction enzyme analysis and Synthego ICE CRISPR web tool, and screening regions of potential off-target genome editing. A genome-edited myoblast cell line with the CHCHD10 c.197G>T mutation corrected, was successfully generated to provide an isogenic control for the patient myoblast cell line. Optimization of myoblast electroporation was successful and conditions used proved to be effective. Clonal cell line creation proved to be challenging with myoblast cells and work is still needed to improve the viability of single-cell clones after FACS. Nevertheless, the advances taken here regarding myoblast genome editing with CRISPR-Cas9 offer a fertile avenue for future research of myoblasts genome manipulation, myogenic disorders, and the role of CHCHD10 in skeletal muscle and SMAJ. Comparing the CHCHD10 protein level and mRNA expression between patient cells, corrected myoblasts, and differentiated myotubes is an area of future research. Future work also includes measuring the mitochondrial integrated stress response in both cell lines and co-culturing myotubes and iPSC derived motor neurons to study the effects of p.G66V on neuromuscular junction (NMJ) formation

The size of the immune repertoire of bacteria

Some bacteria and archaea possess an immune system, based on the CRISPR-Cas mechanism, that confers adaptive immunity against phage. In such species, individual bacteria maintain a "cassette" of viral DNA elements called spacers as a memory of past infections. The typical cassette contains a few dozen spacers. Given that bacteria can have very large genomes, and since having more spacers should confer a better memory, it is puzzling that so little genetic space would be devoted by bacteria to their adaptive immune system. Here, we identify a fundamental trade-off between the size of the bacterial immune repertoire and effectiveness of response to a given threat, and show how this tradeoff imposes a limit on the optimal size of the CRISPR cassette.Comment: 9 pages, 5 figure