scholarly journals Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 628
Author(s):  
Elena McBeath ◽  
Jan Parker-Thornburg ◽  
Yuka Fujii ◽  
Neeraj Aryal ◽  
Chad Smith ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
Genetically Engineered ◽  
Rapid Evaluation ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetically Engineered Mice ◽  
The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

Factor IX: Insights from knock-out and genetically engineered mice

2008 ◽  
Vol 100 (10) ◽  
pp. 563-575 ◽  
Author(s):  
Paul E. Monahan
Keyword(s):  
Mouse Models ◽  
Positional Cloning ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Factor Ix ◽  
Mouse Strains ◽  
Knock Out ◽  
Haemophilia B ◽  
Genetically Engineered Mice ◽  
Knock Out Mice

SummaryThe study of coagulation factors has been rapidly advanced by studies performed in genetically engineered mouse strains. Investigation of factor IX (FIX) has benefited from excellent genedeleted mouse models that recapitulate many of the features of human haemophilia B. Moreover, advanced positional cloning techniques and availability of technology to allow not only knock-out mice, but also knock-in and knock-down mice, provide new opportunities to observe genotype-phenotype and structure-function correlations regarding FIX, as well as the interaction of FIX with inflammatory, immune, and tissue repair systems. In this paper, available FIX knock-out mice and additional haemophilia B mouse models are reviewed specifically in regards to observations these models have facilitated concerning: factor IX gene expression and factor IX protein pharmacokinetics; the role of FIX in haemostasis, thrombosis and wound healing; insights into coagulation FIX arising out of gene therapy applications in haemophilia mouse models; immunology of tolerance or loss of tolerance of FIX and inhibitor antibody formation.

scholarly journals A highly efficient strategy to determine genotypes of genetically-engineered mice using genomic DNA purified from hair roots

2016 ◽  
Vol 51 (2) ◽  
pp. 138-146 ◽  
Author(s):  
Víctor Otaño-Rivera ◽  
Amma Boakye ◽  
Nadja Grobe ◽  
Mohammed M Almutairi ◽  
Shams Kursan ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Genetically Engineered ◽  
Efficient Strategy ◽  
Genomic Dna Isolation ◽  
Hair Roots ◽  
Genetically Engineered Mice ◽  
Purification Methods ◽  
Reducing Costs ◽  
Dna Genotyping

Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.

scholarly journals CRISPR as a Potential Technique to Reduce Suicide Risk

2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Hidemi Zamora ◽  
Javier Cornejo
Keyword(s):  
Cystic Fibrosis ◽  
Suicide Risk ◽  
Gene Editing ◽  
Design Tool ◽  
High Importance ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
The Past ◽  
Rna Design

As suicide is the nineteenth leading cause of death worldwide, it is important to focus on discovering ways to reduce the risk of suicide-related death as much as possible. With CRISPR starting to become increasingly popular over the past few years, this gene editing technique has been used to study how to edit, turn off, or knock out multiple parts of the genome. However, research on genes related to diseases as cystic fibrosis or Alzheimer’s disease has been mainly prioritized and, even though they are of high importance as well, important issues such as suicide have been left into oblivion. Four genes have been proven to be key in influencing suicide risk, showing that not only environmental factors account for an increased possibility of death by this cause. Therefore, gene editing techniques such as CRISPR could be applied in order to knock out those genes and reduce this risk. This research used Synthego’s guide RNA design tool to predict how the use of CRISPR can be helpful in knocking out those four suicide-related genes and, consequently, in preventing suicide. The top-ranked guide RNAs for each gene were used, showing the best results possible and with the least number of off-targets, which, in turn, demonstrates the effectiveness of CRISPR as a potential technique to reduce the number of suicide-related deaths worldwide.

scholarly journals JACKS: joint analysis of CRISPR/Cas9 knock-out screens

2018 ◽  
Author(s):  
Felicity Allen ◽  
Fiona Behan ◽  
Francesco Iorio ◽  
Kosuke Yusa ◽  
Mathew Garnett ◽  
...  
Keyword(s):  
Bayesian Method ◽  
Joint Analysis ◽  
Current Analysis ◽  
Knock Out ◽  
Guide Rna ◽  
Cell Numbers ◽  
Guide Rnas ◽  
Genome Wide ◽  
Fold Reduction ◽  
Hit Identification

Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. Their range of applications remains limited by signal variability from different guide RNAs targeting the same gene, which confounds analysis, and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyses screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification, and allows a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.

scholarly journals Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Satoru Iwata ◽  
Hitomi Nakadai ◽  
Daisuke Fukushi ◽  
Mami Jose ◽  
Miki Nagahara ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genetically Engineered ◽  
Guide Rna ◽  
Chromosomal Inversions ◽  
Genetically Engineered Mice ◽  
Cas9 Protein ◽  
Mouse Zygotes

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells

2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Monika Raab ◽  
Sven Kappel ◽  
Andrea Krämer ◽  
Mourad Sanhaji ◽  
Yves Matthess ◽  
...  
Keyword(s):  
Targeted Therapies ◽  
Mammalian Cells ◽  
Genetically Engineered ◽  
Genetically Engineered Mice

scholarly journals Optical spin-symmetry breaking for high-efficiency directional helicity-multiplexed metaholograms

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Muhammad Ashar Naveed ◽  
Muhammad Afnan Ansari ◽  
Inki Kim ◽  
Trevon Badloe ◽  
Joohoon Kim ◽  
...  
Keyword(s):  
High Efficiency ◽  
Spin Symmetry ◽  
Cost Effective ◽  
Dielectric Materials ◽  
Additional Degree ◽  
Cost Effective Alternative ◽  
Spin Orbit Interactions ◽  
Smart Mobile ◽  
The Cost ◽  
Hydrogenated Amorphous

AbstractHelicity-multiplexed metasurfaces based on symmetric spin–orbit interactions (SOIs) have practical limits because they cannot provide central-symmetric holographic imaging. Asymmetric SOIs can effectively address such limitations, with several exciting applications in various fields ranging from asymmetric data inscription in communications to dual side displays in smart mobile devices. Low-loss dielectric materials provide an excellent platform for realizing such exotic phenomena efficiently. In this paper, we demonstrate an asymmetric SOI-dependent transmission-type metasurface in the visible domain using hydrogenated amorphous silicon (a-Si:H) nanoresonators. The proposed design approach is equipped with an additional degree of freedom in designing bi-directional helicity-multiplexed metasurfaces by breaking the conventional limit imposed by the symmetric SOI in half employment of metasurfaces for one circular handedness. Two on-axis, distinct wavefronts are produced with high transmission efficiencies, demonstrating the concept of asymmetric wavefront generation in two antiparallel directions. Additionally, the CMOS compatibility of a-Si:H makes it a cost-effective alternative to gallium nitride (GaN) and titanium dioxide (TiO2) for visible light. The cost-effective fabrication and simplicity of the proposed design technique provide an excellent candidate for high-efficiency, multifunctional, and chip-integrated demonstration of various phenomena.

scholarly journals Rapid target validation in a Cas9-inducible hiPSC derived kidney model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasaman Shamshirgaran ◽  
Anna Jonebring ◽  
Anna Svensson ◽  
Isabelle Leefa ◽  
Mohammad Bohlooly-Y ◽  
...  
Keyword(s):  
High Efficiency ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Disease Model ◽  
Genetically Engineered ◽  
Model Systems ◽  
Target Validation ◽  
Direct Differentiation ◽  
Kidney Organoids

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

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