De novo PAM generation to reach initially inaccessible target sites for base editing.

Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.

A CRISPR-Cas-associated transposon system for genome editing in Burkholderia cepacia complex species.

Genome editing in non-model bacteria is important to understand gene-to-function links that may differ from those of model microorganisms. Although species of the Burkholderia cepacia complex (Bcc) have great biotechnological capacities, the limited genetic tools available to understand and mitigate their pathogenic potential hamper their utilization in industrial applications. To broaden the genetic tools available for Bcc species, we developed RhaCAST, a targeted DNA insertion platform based on a CRISPR-associated transposase driven by a rhamnose-inducible promoter. We demonstrated the utility of the system for targeted insertional mutagenesis in the Bcc strains B. cenocepacia K56-2 and Burkholderia multivorans ATCC17616. We showed that the RhaCAST system can be used for loss- and gain-of-function applications. Importantly, the selection marker could be excised and reused to allow iterative genetic manipulation. The RhaCAST system is faster, easier, and more adaptable than previous insertional mutagenesis tools available for Bcc species and may be used to disrupt pathogenicity elements and insert relevant genetic modules, enabling Bcc biotechnological applications. IMPORTANCE: Species of the Burkholderia cepacia complex (Bcc) have great biotechnological potential but are also opportunistic pathogens. Genetic manipulation of Bcc species is necessary to understand gene-to-function links. However, limited genetic tools are available to manipulate Bcc, hindering our understanding of their pathogenic traits and their potential in biotechnological applications. We developed a genetic tool based on CRISPR-associated transposase to increase the genetic tools available for Bcc species. The genetic tool we developed in this study can be used for loss and gain of function in Bcc species. The significance of our work is in expanding currently available tools to manipulate Bcc.

WAX INDUCER 1 Regulates ß-Diketone Biosynthesis by Mediating Expression of the Cer-cqu Gene Cluster in Barley.

Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with ß-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the ß-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.

GMO Genetic Elements Thesaurus (GMO-GET): a controlled vocabulary for the consensus designation of introduced or modified genetic elements in genetically modified organisms.

BACKGROUND: Various databases on genetically modified organisms (GMOs) exist, all with their specific focus to facilitate access to information needed for, e. g., the assistance in risk assessment, the development of detection and identification strategies or inspection and control activities. Each database has its unique approach towards the subject. Often these databases use different terminology to describe the GMOs. For adequate GMO addressing and identification and exchange of GMO-related information it is necessary to use commonly agreed upon concepts and terminology. RESULT: A hierarchically structured controlled vocabulary describing the genetic elements inserted into conventional GMOs, and GMOs developed by the use of gen(om)e-editing is presented: the GMO genetic element thesaurus (GMO-GET). GMO-GET can be used for GMO-related documentation, including GMO-related databases. It has initially been developed on the basis of two GMO databases, i.e. the Biosafety Clearing-House and the EUginius database. CONCLUSION: The use of GMO-GET will enable consistent and compatible information (harmonisation), also allowing an accurate exchange of information between the different data systems and thereby facilitating their interoperability. GMO-GET can also be used to describe genetic elements that are altered in organisms obtained through current targeted genome-editing techniques.

A Simple Heat Treatment Increases SpCas9-Mediated Mutation Efficiency in Arabidopsis.

The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37°C for 24 h increased the mutation efficiency at all four target sites from 3 to 42%, 43 to 62%, 54 to 75% and 89 to 91%, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37°C for 24 h as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells.

Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.

Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8'OH1 loci.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts.

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33-152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Highly Efficient Temperature Inducible CRISPR-Cas9 Gene Targeting in Drosophila suzukii.

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene.

BACKGROUND: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. RESULTS: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. CONCLUSIONS: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high ß-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew.

BACKGROUND: The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). RESULTS: To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. CONCLUSION: CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.

First-generation genome editing in potato using hairy root transformation.

Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening.

Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.

Converting a ß-N-acetylhexosaminidase into two trans-ß-N-acetylhexosaminidases by domain-targeted mutagenesis.

We have recently derived a ß-N-acetylhexosaminidase, BbhI, from Bifidobacterium bifidum JCM 1254, which could regioselectively synthesize GlcNAcß1-3Galß1-4Glc with a yield of 44.9%. Here, directed evolution of BbhI by domain-targeted mutagenesis was carried out. Firstly, the GH20 domain was selected for random mutagenesis using MEGAWHOP method and a small library of 1300 clones was created. A total of 734 colonies with reduced hydrolytic activity were isolated, and three mutants with elevated transglycosylation yields, GlcNAcß1-3Galß1-4Glc yields of 68.5%, 74.7%, and 81.1%, respectively, were obtained. Subsequently, nineteen independent mutants were constructed according to all the mutation sites in these three mutants. After transglycosylation analysis, Asp714 and Trp773 were identified as key residues for improvement in transglycosylation ability and were chosen for the second round of directed evolution by site-saturation mutagenesis. Two most efficient mutants D714T and W773R that acted as trans-ß-N-acetylhexosaminidase were finally achieved. D714T with the substitution at the putative nucleophile assistant residue Asp714 by threonine showed high yield of 84.7% with unobserved hydrolysis towards transglycosylation product. W773R with arginine substitution at Trp773 residue locating at the entrance of catalytic cavity led to the yield up to 81.8%. The kcat/Km values of D714T and W773R for hydrolysis of pNP-ß-GlcNAc displayed drastic decreases. NMR investigation of protein-substrate interaction revealed an invariable mode of the catalytic cavity of D714T, W773R, and WT BbhI. The collective motions of protein model showed the mutations Thr714 and Arg773 exerted little effect on the dynamics of the inside but a large effect on the dynamics of the outside of catalytic cavity.

Prion gene paralogs are dispensable for early zebrafish development and have nonadditive roles in seizure susceptibility.

Normally folded prion protein (PrPC) and its functions in healthy brains remain underappreciated compared with the intense study of its misfolded forms ("prions," PrPSc) during the pathobiology of prion diseases. This impedes the development of therapeutic strategies in Alzheimer's and prion diseases. Disrupting the zebrafish homologs of PrPC has provided novel insights; however, mutagenesis of the zebrafish paralog prp2 did not recapitulate previous dramatic developmental phenotypes, suggesting redundancy with the prp1 paralog. Here, we generated zebrafish prp1 loss-of-function mutant alleles and dual prp1-/-;prp2-/- mutants. Zebrafish prp1-/- and dual prp1-/-;prp2-/- mutants resemble mammalian Prnp knockouts insofar as they lack overt phenotypes, which surprisingly contrasts with reports of severe developmental phenotypes when either prp1 or prp2 is knocked down acutely. Previous studies suggest that PrPC participates in neural cell development/adhesion, including in zebrafish where loss of prp2 affects adhesion and deposition patterns of lateral line neuromasts. In contrast with the expectation that prp1's functions would be redundant to prp2, they appear to have opposing functions in lateral line neurodevelopment. Similarly, loss of prp1 blunted the seizure susceptibility phenotypes observed in prp2 mutants, contrasting the expected exacerbation of phenotypes if these prion gene paralogs were serving redundant roles. In summary, prion mutant fish lack the overt phenotypes previously predicted, and instead they have subtle phenotypes similar to mammals. No evidence was found for functional redundancy in the zebrafish prion gene paralogs, and the phenotypes observed when each gene is disrupted individually are consistent with ancient functions of prion proteins in neurodevelopment and modulation of neural activity.

Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staphylococcus aureus.

Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a double-stranded break (DSB) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single-stranded break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5' or 3' overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell-specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will.

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.

CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max.L).

BACKGROUND: Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis. RESULTS: Here we demonstrated an efficient sequence specific integration/mutation of FAD2-2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2-2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2-2 loci. The integration of FAD2-2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis. CONCLUSION: High frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.

Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex.

The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.