scholarly journals A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila

2014 ◽  
Author(s):  
Xu Zhang ◽  
Wouter Koolhaas ◽  
Frank Schnorrer
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Model Organisms ◽  
Second Step ◽  
Fourth Chromosome ◽  
Gene Modification ◽  
Experimental Conditions ◽  
Cassette Exchange ◽  
Quantum Leap ◽  
Universal Applicability

The development of CRISPR/Cas9 technologies promises a quantum leap in genome-engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila are still restricted to a few genes, use variable experimental conditions and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient, two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different loci, including a gene on the fourth chromosome, at comparably high efficiencies, suggesting that any fly lab can engineer their favourite gene for a broad range of applications within about three months.

scholarly journals TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 19-40 ◽  
Author(s):  
A. A. Nemudryi ◽  
K. R. Valetdinova ◽  
S. P. Medvedev ◽  
S. M. Zakian
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
Disease Modeling ◽  
Regulation Of Gene Expression ◽  
Hereditary Disease ◽  
Transcription Activator ◽  
Wide Range ◽  
High Standards ◽  
Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

CRISPR-Based Tools in Immunity

2019 ◽  
Vol 37 (1) ◽  
pp. 571-597 ◽  
Author(s):  
Dimitre R. Simeonov ◽  
Alexander Marson
Keyword(s):  
Genome Engineering ◽  
Disease Risk ◽  
Ease Of Use ◽  
Model Organisms ◽  
Mechanistic Studies ◽  
Genetic Circuits ◽  
New Era ◽  
Wide Range ◽  
Health And Disease ◽  
Cancer Immunotherapies

CRISPR technology has opened a new era of genome interrogation and genome engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the CRISPR system has been repurposed as an adaptable tool for genome editing and multiple other applications. CRISPR's ease of use, precision, and versatility have led to its widespread adoption, accelerating biomedical research and discovery in human cells and model organisms. Here we review CRISPR-based tools and discuss how they are being applied to decode the genetic circuits that control immune function in health and disease. Genetic variation in immune cells can affect autoimmune disease risk, infectious disease pathogenesis, and cancer immunotherapies. CRISPR provides unprecedented opportunities for functional mechanistic studies of coding and noncoding genome sequence function in immunity. Finally, we discuss the potential of CRISPR technology to engineer synthetic cellular immunotherapies for a wide range of human diseases.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

scholarly journals Resurrecting phenotypes from ancient DNA sequences: promises and perspectives

2015 ◽  
Vol 93 (9) ◽  
pp. 701-710 ◽  
Author(s):  
K.L. Campbell ◽  
M. Hofreiter
Keyword(s):  
Ancient Dna ◽  
Dna Sequences ◽  
Cell Biology ◽  
Genome Engineering ◽  
Ectopic Expression ◽  
Evolutionary Transitions ◽  
Extinct Species ◽  
Evolutionary Origins ◽  
Structural And Functional Properties

Anatomical changes in extinct mammalian lineages over evolutionary time, such as the loss of fingers and teeth and the rapid increase in body size that accompanied the late Miocene dispersal of the progenitors of Steller’s sea cows (Hydrodamalis gigas (Zimmermann, 1780)) into North Pacific waters and the convergent development of a thick pelage and accompanying reductions in ear and tail surface area of woolly mammoths (Mammuthus primigenius (Blumenbach, 1799)) and woolly rhinoceros (Coelodonta antiquitatis (Blumenbach, 1799)), are prime examples of adaptive evolution underlying the exploitation of new habitats. It is likely, however, that biochemical specializations adopted during these evolutionary transitions were of similar or even greater biological importance. As these “living” processes do not fossilize, direct information regarding the physiological attributes of extinct species has largely remained beyond the range of scientific inquiry. However, the ability to retrieve genomic sequences from ancient DNA samples, combined with ectopic expression systems, now permit the evolutionary origins and structural and functional properties of authentic prehistoric proteins to be examined in great detail. Exponential technical advances in ancient DNA retrieval, enrichment, and sequencing will soon permit targeted generation of complete genomes from hundreds of extinct species across the last one million years that, in combination with emerging in vitro expression, genome engineering, and cell differentiation techniques, promises to herald an exciting new trajectory of evolutionary research at the interface of biochemistry, genomics, palaeontology, and cell biology.

Low prevalence of Chlamydia psittaci infection in French patients with ocular adnexal lymphomas (OAL)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 17536-17536
Author(s):  
D. Decaudin ◽  
A. Subtil ◽  
A. Ferreri ◽  
A. Vincent-Salomon ◽  
M. Ponzoni ◽  
...  
Keyword(s):  
B Cell ◽  
Dna Sequences ◽  
Mononuclear Cells ◽  
Lymphoid Hyperplasia ◽  
Lymphoproliferative Disease ◽  
Chlamydia Psittaci ◽  
Reactive Lymphoid Hyperplasia ◽  
Experimental Conditions ◽  
Peripheral Blood Mononuclear ◽  
Geographical Variations

17536 Background: A high prevalence of Chlamydia psittaci infection in both tumor tissues and peripheral blood mononuclear cells of Italian patients with OAL has been reported (Ferreri AJ, Guidoboni M, Ponzoni M, De Conciliis C, Dell’Oro S, Fleischhauer K, et al. Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J Natl Cancer Inst 2004;96(8):586–94.) This association has not been confirmed in some regions of the USA, and no data from other European countries are available. We therefore investigated the presence of DNA of C. psittaci, C. trachomatis, and C. pneumoniae DNA in French patients (pts) with OAL. Methods: Tumor samples from ophthalmologic biopsies of 16 OAL pts (10 conjunctiva, 6 orbit) were included in the study. Histologic type were MALT-type (n = 8), lymphoplasmocytic (n = 6), follicular (n = 1), and diffuse large B-cell (n = 1) lymphomas. Two other groups of lymphoproliferative disease were analyzed as controls. First, ten cases of nodal lymphomas (follicular and marginal zone B-cell lymphomas), and ten cases of reactive lymphoid hyperplasia were analyzed. A multiplex touchdown, enzyme time-release PCR designed to simultaneously detect C. psittaci, C. pneumoniae and C. trachomatis DNA sequences was performed. PCR analyses were performed in duplicate in an independent setting either inat the Curie Institut Curie in e, Paris, and in the National Cancer Institute, Aviano. Results: DNA of C. psittaci DNA was detected in the tumoral tissue of only one patient with follicular OAL. No DNA sequences of C. psittaci, C. pneumoniae and C. trachomatis DNA sequences were was detected in all any of the other OALs, or controls. Conclusions: The prevalence of C. psittaci infection in French pts with OAL was sensibly significantly lower than that reported in Italian series. Cross-controls between the two laboratories indicate that this finding is not due to different experimental conditions. Discrepancies may be explained by an heterogeneous epidemiological distribution of the bacterial infection. Large studies aimed to investigate geographical variations in the prevalence of this association are warranted. No significant financial relationships to disclose.

scholarly journals Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

1994 ◽  
Vol 14 (10) ◽  
pp. 6936-6943 ◽  
Author(s):  
P J Detloff ◽  
J Lewis ◽  
S W John ◽  
W R Shehee ◽  
R Langenbach ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Second Step ◽  
Globin Genes ◽  
Beta Globin ◽  
Es Cell ◽  
Beta Globin Gene ◽  
Targeting Strategy

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

scholarly journals A Cas9 with Complete PAM Recognition for Adenine Dinucleotides

2018 ◽  
Author(s):  
Noah Jakimo ◽  
Pranam Chatterjee ◽  
Lisa Nip ◽  
Joseph M Jacobson
Keyword(s):  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Genome Engineering ◽  
Protein Domains ◽  
Human Cells ◽  
Gene Modification ◽  
Editing Efficiency ◽  
Base Editing ◽  
Protospacer Adjacent Motif

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a Type-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased” nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.

scholarly journals The Rise of “Digital Biology”: We need not only open, FAIR but also sustainable data!

2019 ◽  
Vol 3 ◽  
Author(s):  
Philippe Grandcolas
Keyword(s):  
At Risk ◽  
Dna Sequences ◽  
Life Sciences ◽  
Digital Data ◽  
Model Organisms ◽  
Biological Knowledge ◽  
Selected Lines ◽  
Taxonomic Descriptions ◽  
Global Coherence ◽  
Occurrence Records

Biology has already experienced great divides that decreased its global coherence and its ability to answer important scientific and societal concerns. For example in the XXth century, the so-called “Life Sciences” developed remarkably in comparison to Natural History sciences. This way, the approaches on model organisms dominated or prevented other approaches from being carried out on more diverse organisms, which may have given a misleading feeling of generality for the results obtained. Another great divide is at risk of developing now with the rise of what could be called “Digital Biology,” separating from other “material-based” approaches in its tendency to consider digital data only. Some biologists adopt a somewhat essentialist view of species and DNA, considering that enough knowledge is now accumulated, and that species records can be kept and saved as digital data only (Grandcolas 2017). Examples of this include occurrence records without specimens or auxiliary documents, taxonomic descriptions based on photographs, DNA sequences without vouchers, and, lastly, DNA sequences without taxonomic names. This tendency puts at risk the sustainability, growth, and coherence of biological knowledge that is organized in a system wherein all data and notions are connected via specimens, with names and sequences being a retrieval means (Troudet et al. 2018). This tendency also ignores the robust foundation of biology, the data of which are linked to collections, vouchers, and stocks. The foundation of physical specimens exists for data concerning any live beings, be they rare wild species or selected lines of model organisms. There are now many calls for open and FAIR science, with results, methods, tools, and data not only findable, accessible, and interoperable but also re-usable. More than FAIR and digitally re-usable, data need to be sustainable. It is needed that their meaning and significance can be re-analysed, re-interpreted by going back as far as possible to material vouchers. We urge then scientists to consider this question by providing all necessary material elements to make open and FAIR data sustainable as well.

scholarly journals Genome-engineering with CRISPR-Cas9 in the mosquitoAedes aegypti

2014 ◽  
Author(s):  
Kathryn E Kistler ◽  
Leslie B Vosshall ◽  
Benjamin J Matthews
Keyword(s):  
Genome Engineering ◽  
Germ Line ◽  
Dna Binding Protein ◽  
Model Organisms ◽  
Homing Endonucleases ◽  
Loss Of Function ◽  
Dna Base ◽  
Different Types ◽  
Flexible Genome ◽  
Repair Mechanisms

The mosquitoAedes aegyptiis a potent vector of the Chikungunya, yellow fever, and Dengue viruses, which result in hundreds of millions of infections and over 50,000 human deaths per year. Loss-of-function mutagenesis inAe. aegyptihas been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to generate target specificity for a particular stretch of genomic DNA. Here, we describe the first use of the CRISPR-Cas9 system to generate targeted, site-specific mutations inAe. aegypti. CRISPR-Cas9 relies on RNA-DNA base-pairing to generate targeting specificity, resulting in cheaper, faster, and more flexible genome-editing reagents. We investigate the efficiency of reagent concentrations and compositions, demonstrate the ability of CRISPR-Cas9 to generate several different types of mutations via disparate repair mechanisms, and show that stable germ-line mutations can be readily generated at the vast majority of genomic loci tested. This work offers a detailed exploration into the optimal use of CRISPR-Cas9 inAe. aegyptithat should be applicable to non-model organisms previously out of reach of genetic modification.

scholarly journals Cre-assisted Fine-mapping of Neural Circuits using Orthogonal Split Inteins

2019 ◽  
Author(s):  
Haojiang Luan ◽  
Alexander Kuzin ◽  
Ward F. Odenwald ◽  
Benjamin H. White
Keyword(s):  
Neural Circuits ◽  
Genetic Model ◽  
Model Organisms ◽  
Second Step ◽  
Developmental Origins ◽  
Combinatorial Method ◽  
Brain Circuits ◽  
Alternative Techniques ◽  
The Brain

Summary:Genetic methods for targeting small numbers of neurons of a specific type are critical for mapping the brain circuits underlying behavior. Existing methods can provide exquisite targeting precision in favorable cases, but for many cases alternative techniques will be required. Here, we introduce a new step-wise combinatorial method for sequentially refining neuronal targeting: Depending on the restriction achieved at the first step, a second step can be easily implemented to further refine expression. For both steps, the new method relies on two independent intersections. The primary intersection targets neurons based on their developmental origins (i.e. lineage) and terminal identities, while the second intersection limits the number of lineages represented in the primary intersection by selecting lineages with overlapping activity of two distinct enhancers during neurogenesis. Our method relies critically on two libraries of 134 transgenic fly lines that express fragments of a split Cre recombinase under the control of distinct neuroblast enhancers. The split Cre fragments are fused to non-interacting pairs of split inteins, which ensure reconstitution of full-length and active Cre when all fragments are expressed in the same cell. Our split Cre system, together with its open source libraries, represent off-the-shelf components that should facilitate the targeting and characterization of brain circuits in Drosophila. Our methodology may also prove useful in other genetic model organisms.

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