scholarly journals An optimized CRISPR/Cas toolbox for efficient germline and somatic genome engineering in Drosophila

2014 ◽  
Author(s):  
Fillip Port ◽  
Hui-Min Chen ◽  
Tzumin Lee ◽  
Simon L Bullock
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Systematic Evaluation ◽  
Loss Of Function ◽  
Potent Effect ◽  
Single Nucleotide ◽  
Guide Rna ◽  
U6 Snrna ◽  
Somatic Genome ◽  
Mutant Phenotypes

The type II CRISPR/Cas system has recently emerged as a powerful method to manipulate the genomes of various organisms. Here, we report a novel toolbox for high efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germline restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. Choosing an appropriate combination of Cas9 and gRNA allows targeting of essential and non-essential genes with transmission rates ranging from 25% - 100%. We also provide evidence that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis and, in combination with oligonucleotide donors, to precisely edit the genome by homologous recombination with efficiencies that do not require the use of visible markers. Lastly, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. In summary, our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single nucleotide precision. Our results also pave the way for high throughput genetic screening with CRISPR/Cas.

scholarly journals Plasmid-based and -free methods using CRISPR/Cas9 system for replacement of targeted genes in Colletotrichum sansevieriae

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Masayuki Nakamura ◽  
Yuta Okamura ◽  
Hisashi Iwai
Keyword(s):  
High Efficiency ◽  
Pathogenic Fungus ◽  
Gene Replacement ◽  
Gene Encoding ◽  
Melanin Biosynthesis ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
U6 Snrna ◽  
Cas9 Protein ◽  
Scytalone Dehydratase

AbstractThe CRISPR-Cas9 system has a potential for wide application in organisms that particularly present low homologous integration rates. In this study, we developed three different methods using this system to replace a gene through homology-directed repair in the plant pathogenic fungus Colletotrichum sansevieriae, which has a low recombination frequency. The gene encoding scytalone dehydratase was used as the target so that mutants can be readily distinguished owning to a lack of melanin biosynthesis. First, we performed a plasmid-based method using plasmids containing a Cas9 expression cassette and/or a single-guide RNA (sgRNA) under the control of the endogenous U6 snRNA promoter, and 67 out of 69 (97.1%) transformants exhibited a melanin-deficient phenotype with high efficiency. Second, we performed a transformation using a Cas9 protein/sgRNA complex and obtained 23 out of 28 (82.1%) transformants. Lastly, we developed a hybrid system combining a Cas9 protein and donor DNA-sgRNA expression plasmid, which yielded 75 out of 84 (89.2%) transformants. This system was also applicable to four other genes at different loci of the fungus. This is the first study to establish a CRISPR/Cas9 gene replacement system in Colletotrichum spp. and it presents a potential application for a broad range of use in other species of the genus.

scholarly journals A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yaojun Tong ◽  
Tue S. Jørgensen ◽  
Christopher M. Whitford ◽  
Tilmann Weber ◽  
Sang Yup Lee
Keyword(s):  
Genome Engineering ◽  
Genetic Manipulation ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
E Coli ◽  
Potential Basis ◽  
Low Efficiency ◽  
Nucleotide Resolution

AbstractCRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

scholarly journals Transgene-mediated skeletal phenotypic variation in zebrafish

2019 ◽  
Author(s):  
Charles B. Kimmel ◽  
Alexander L. Wind ◽  
Whitney Oliva ◽  
Samuel D. Ahlquist ◽  
Charline Walker ◽  
...  
Keyword(s):  
Transposable Elements ◽  
High Efficiency ◽  
Single Copy ◽  
The United States ◽  
Biological Action ◽  
Genetically Engineered ◽  
Biological Research ◽  
Loss Of Function ◽  
Phenotypic Effect ◽  
Mutant Phenotypes

AbstractWhen considering relationships between genotype and phenotype we frequently ignore the fact that the genome of a typical animal, notably including that of a fish and a human, harbors a huge amount of foreign DNA. Some of it, including the DNA of “autonomous” transposable elements, can spontaneously mobilize to occupy new chromosomal sites and take on new functions, presenting a challenge to the host organism and also possibly introducing new fuel for evolutionary change. Transposable elements are useful for introducing transgenes, integrating them into host genomes with high efficiency. Transgenesis has become very widespread in biological research, and in our society at large. This year the governments of both Canada and the United States have approved the first use of ‘genetically engineered’ animals in food production, Atlantic salmon, Salmo salar. With the recent advent of amazing gene-editing technology, there is no doubt that the transgene industry will grow explosively in the coming years. The biology of transgenes needs to be included in our understanding of the genome. It is in this spirit that we have investigated an unexpected and novel phenotypic effect of the chromosomally integrated transgene fli1a-F-hsp70l:Gal4VP16. We examine larval fras1 mutant zebrafish (Danio rerio). Gal4VP16 is a potent transcriptional activator, and already well known for toxicity and mediating unusual transcriptional effects. In the presence of the transgene, phenotypes in the neural crest-derived craniofacial skeleton, notably fusions and shape changes associated with loss of function fras1 mutations, are made more severe, as we quantify by scoring phenotypic penetrance, the fraction of mutants expressing the trait. A very interesting feature is that the enhancements are highly specific for fras1 mutant phenotypes – occurring in the apparent absence of more wide-spread changes. Except for the features due to the fras1 mutation, the transgene-bearing larvae appear generally healthy and to be developing normally. The transgene behaves as a genetic partial dominant: A single copy is sufficient for the enhancements, yet, for some traits, two copies may exert a stronger effect. We made new strains bearing independent insertions of the fli1a-F-hsp70l:Gal4VP16 transgene in new locations in the genome, and observed increased severities of the same phenotypes as observed for the original insertion. This finding suggests that sequences within the transgene, e.g. Gal4VP16, are responsible for the enhancements, rather than effect on neighboring host sequences (such as an insertional mutation). The specificity, and biological action underlying the traits, are subjects of considerable interest for further investigation, as we discuss. Our findings show that work with transgenes needs to be undertaken with caution and attention to detail.

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 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 Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Steven Lin ◽  
Brett T Staahl ◽  
Ravi K Alla ◽  
Jennifer A Doudna
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Embryonic Stem ◽  
Human Cells ◽  
Dna Breaks ◽  
Site Specific ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Cell Mortality

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

scholarly journals Various Aspects of a Gene Editing System—CRISPR–Cas9

2020 ◽  
Vol 21 (24) ◽  
pp. 9604
Author(s):  
Edyta Janik ◽  
Marcin Niemcewicz ◽  
Michal Ceremuga ◽  
Lukasz Krzowski ◽  
Joanna Saluk-Bijak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Adaptive Immune ◽  
Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

scholarly journals CRISPR RNA-guided integrases for high-efficiency and multiplexed bacterial genome engineering

2020 ◽  
Author(s):  
Phuc Leo H. Vo ◽  
Carlotta Ronda ◽  
Sanne E. Klompe ◽  
Ethan E. Chen ◽  
Christopher Acree ◽  
...  
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Computational Algorithm ◽  
Bacterial Genome ◽  
Expression Vectors ◽  
Type I ◽  
Dna Integration ◽  
Guide Rna ◽  
Marker Free ◽  
Rna Design

Tn7-like transposons are pervasive mobile genetic elements in bacteria that mobilize using heteromeric transposase complexes comprising distinct targeting modules. We recently described a Tn7-like transposon from Vibrio cholerae that employs a Type I-F CRISPR–Cas system for RNA-guided transposition, in which Cascade directly recruits transposition proteins to integrate donor DNA downstream of genomic target sites complementary to CRISPR RNA. However, the requirement for multiple expression vectors and low overall integration efficiencies, particularly for large genetic payloads, hindered the practical utility of the transposon. Here, we present a significantly improved INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting) system for targeted, multiplexed, and marker-free DNA integration of up to 10 kilobases at ~100% efficiency. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplex insertions in three genomic loci, and facile multi-loci deletions when combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in other biomedically- and industrially-relevant bacteria, and developed an accessible computational algorithm for guide RNA design. This work establishes INTEGRATE as a versatile and portable tool that enables multiplex and kilobase-scale genome engineering.

scholarly journals Optimizing Systems for Cas9 Expression in Toxoplasma gondii

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Benedikt M. Markus ◽  
George W. Bell ◽  
Hernan A. Lorenzi ◽  
Sebastian Lourido
Keyword(s):  
Toxoplasma Gondii ◽  
Genome Engineering ◽  
Childhood Mortality ◽  
Loss Of Function ◽  
Content Type ◽  
Guide Rna ◽  
Severe Diarrhea ◽  
Biological Studies ◽  
Recent Developments ◽  
Improved Stability

ABSTRACT CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression. IMPORTANCE Toxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii. As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species.

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.

Sign in / Sign up

Export Citation Format

Share Document