scholarly journals Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

2015 ◽  
Vol 112 (11) ◽  
pp. 3570-3575 ◽  
Author(s):  
Kabin Xie ◽  
Bastian Minkenberg ◽  
Yinong Yang
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Crop Improvement ◽  
Processing System ◽  
Basic Research ◽  
Molecular Therapy ◽  
General Strategy ◽  
Trna Processing ◽  
Guide Rna

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

scholarly journals High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Weiwei Qi ◽  
Tong Zhu ◽  
Zhongrui Tian ◽  
Chaobin Li ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Gene Editing ◽  
High Efficiency ◽  
Processing System ◽  
Trna Processing

scholarly journals All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

2018 ◽  
Author(s):  
Raed Ibraheim ◽  
Chun-Qing Song ◽  
Aamir Mir ◽  
Nadia Amrani ◽  
Wen Xue ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neisseria Meningitidis ◽  
Genome Editing ◽  
Viral Vectors ◽  
Genome Engineering ◽  
Degradation Pathway ◽  
Type I ◽  
Guide Rna ◽  
Hereditary Tyrosinemia

AbstractClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.

scholarly journals Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

2018 ◽  
Author(s):  
Wenqiang Li ◽  
Shuntang Li ◽  
Jie Qiao ◽  
Fei Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
General Strategy ◽  
Dna Assembly ◽  
E Coli ◽  
Assembly Method ◽  
Direct Preparation

AbstractCRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

scholarly journals A potential clue for the application of prime editing in tomato, a dicot plant

2021 ◽  
Author(s):  
Tien Van Vu ◽  
Jihae Kim ◽  
Swati Das ◽  
Jae-Yean Kim
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Plant Genome ◽  
Guide Rna ◽  
Genome Modification ◽  
Potential Applications ◽  
Targeted Deep Sequencing ◽  
Synthetic Sequence

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a priming extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. However, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. However, our data obtained by targeted deep sequencing also revealed inefficient PE activity in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. These data could also help explain the recent successes of some prime editors in plants using improved expression systems. Our work provides an important clue for the application of the PE approach in crop improvement.

scholarly journals Efficient targeted integration directed by short homology in zebrafish and mammalian cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Wesley A Wierson ◽  
Jordan M Welker ◽  
Maira P Almeida ◽  
Carla M Mann ◽  
Dennis A Webster ◽  
...  
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Cost Effective ◽  
Strand Break ◽  
End Joining ◽  
Cost Effective Approach ◽  
Targeted Integration

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

scholarly journals New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Valmik K. Vyas ◽  
G. Guy Bushkin ◽  
Douglas A. Bernstein ◽  
Matthew A. Getz ◽  
Magdalena Sewastianik ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Genome Engineering ◽  
High Efficiency ◽  
Selection Marker ◽  
Important Species ◽  
Content Type ◽  
Guide Rna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
The Creation

ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans , Saccharomyces cerevisiae , Candida glabrata , and Naumovozyma castellii . These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata , as well as in the related yeasts S. cerevisiae and N. castellii . The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

scholarly journals CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research

2015 ◽  
Author(s):  
Fillip Port ◽  
Nadine Muschalik ◽  
Simon L Bullock
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Basic Research ◽  
Evidence Based ◽  
Gene Drive ◽  
Highly Active ◽  
Genome Modification ◽  
Target Sites ◽  
Gene Drives

CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. CRISPR components produced by integrated transgenes have been shown to mutagenise some genomic target sites in Drosophila melanogaster with high efficiency, but whether this is a general feature of this system remains unknown. Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.

scholarly journals GeneWeld: a method for efficient targeted integration directed by short homology

2018 ◽  
Author(s):  
Wesley A. Wierson ◽  
Jordan M. Welker ◽  
Maira P. Almeida ◽  
Carla M. Mann ◽  
Dennis A. Webster ◽  
...  
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
High Specificity ◽  
Strand Break ◽  
Double Strand Break ◽  
Genomic Locus ◽  
Multiple Loci ◽  
Integration Strategy ◽  
Targeted Integration

AbstractChoices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids forGeneTagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates thatin vivotargeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand breakin vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

scholarly journals Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daichi G. Suzuki ◽  
Hiroshi Wada ◽  
Shin-ichi Higashijima
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Evolutionary Developmental Biology ◽  
Model Organisms ◽  
Donor Plasmid ◽  
Guide Rna ◽  
Research Fields ◽  
Design Flexibility ◽  
Genetic Techniques ◽  
Mrna Targeting

AbstractThe lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.

scholarly journals Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo

2017 ◽  
Author(s):  
Ruth M Williams ◽  
Upeka Senanayake ◽  
Mara Artibani ◽  
Gunes Taylor ◽  
Daniel Wells ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Neural Crest ◽  
Chicken Embryo ◽  
Genome Engineering ◽  
Next Generation ◽  
Guide Rna ◽  
Regulatory Interactions ◽  
Gene Regulatory ◽  
Generation Sequencing

AbstractCRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present a highly efficient toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise efficient guide-RNA expression from novel chick U6-mini-vectors, provide a strategy for rapid somatic gene knockout and establish protocol for evaluation of mutational penetrance by targeted next generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.Summary StatementWe present an optimised toolkit for efficient genome and epigenome engineering using CRISPR in chicken embryos, with a particular focus on probing gene regulatory interactions during neural crest development.List of AbbreviationsGenome Engineering (GE), Epigenome Engineering (EGE), single guide RNA (sgRNA), Neural Crest (NC), Transcription Factor (TF), Next Generation Sequencing (NGS), somite stage (ss), Hamburger Hamilton (HH).

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