scholarly journals Construction and identification of Rab7-ROSA26 knock-in mice by CRISPR/Cas9 technology

2020 ◽  
Author(s):  
Qing Xu ◽  
Linfang Jin ◽  
Xiaowei Qi
Keyword(s):  
Genome Editing ◽  
Southern Blot ◽  
Renal Fibrosis ◽  
Gene Knockout ◽  
Gene Insertion ◽  
System A ◽  
A Genome ◽  
The Relationship

Abstract Background: CRISPR/Cas9 system, a Genome editing technique, can achieve the purpose of gene knockout and gene insertion. Because we want to study the relationship between Rab7 and renal fibrosis in vivo, we should construct Rab7-knock-in mice. Results: We constructed Rab7-knock-in mice by CRISPR/Cas9 technology. Using the identification of PCR, southern blot and sequencing, the detected results were in line with our expectation. Conclusions: We constructed Rab7-knock-in mice successful, which can be provided for studying the relationship between Rab7 and renal fibrosis.

scholarly journals ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Camilo Breton ◽  
Peter M. Clark ◽  
Lili Wang ◽  
Jenny A. Greig ◽  
James M. Wilson
Keyword(s):  
Next Generation Sequencing ◽  
Genome Editing ◽  
Next Generation ◽  
Aav Vector ◽  
Genome Wide ◽  
A Genome ◽  
Vector Sequences ◽  
Target Activity ◽  
Generation Sequencing

Abstract Background Identifying nuclease-induced double-stranded breaks in DNA on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies. We previously demonstrated that after administering adeno-associated viral (AAV) vector-mediated genome-editing strategies in vivo, vector sequences integrated into the host organism’s genomic DNA at double-stranded breaks. Thus, identifying the genomic location of inserted AAV sequences would enable us to identify DSB events, mainly derived from the nuclease on- and off-target activity. Results Here, we developed a next-generation sequencing assay that detects insertions of specific AAV vector sequences called inverted terminal repeats (ITRs). This assay, ITR-Seq, enables us to identify off-target nuclease activity in vivo. Using ITR-Seq, we analyzed liver DNA samples of rhesus macaques treated with AAV vectors expressing a meganuclease. We found dose-dependent off-target activity and reductions in off-target events induced by further meganuclease development. In mice, we identified the genomic locations of ITR integration after treatment with Cas9 nucleases and their corresponding single-guide RNAs. Conclusions In sum, ITR-Seq is a powerful method for identifying off-target sequences induced by AAV vector-delivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genome-editing nucleases in animal models and clinical studies. This information can help enhance the safety profile of gene-editing therapies.

scholarly journals A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

2017 ◽  
Vol 4 (5) ◽  
pp. 170095 ◽  
Author(s):  
Tom Beneke ◽  
Ross Madden ◽  
Laura Makin ◽  
Jessica Valli ◽  
Jack Sunter ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Gene Knockout ◽  
Guide Rna ◽  
Functional Studies

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

scholarly journals Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it’s Insertion into genomic safe harbor AAVS1 site using CRISPR–Cas9

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1498
Author(s):  
Nisha Vats ◽  
Madhusudana Girija Sanal ◽  
Senthil Kumar Venugopal ◽  
Pankaj Taneja ◽  
Shiv Kumar Sarin
Keyword(s):  
Lipid A ◽  
Safe Harbor ◽  
Adeno Associated Virus ◽  
E Coli ◽  
Gene Insertion ◽  
Genomic Location ◽  
A Genome ◽  
Bile Acid Transporter

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man.  Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool.  We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

scholarly journals Precise removal of Calm1 long 3′ UTR isoform by CRISPR-Cas9 genome editing impairs dorsal root ganglion development in mice

2019 ◽  
Author(s):  
Hannah N. Gruner ◽  
Bongmin Bae ◽  
Maebh Lynch ◽  
Daniel Oliver ◽  
Kevin So ◽  
...  
Keyword(s):  
Dorsal Root Ganglion ◽  
Genome Editing ◽  
Neural Development ◽  
Dorsal Root ◽  
Physiological Role ◽  
Mrna Isoforms ◽  
A Genome ◽  
Mouse Tissues ◽  
Cleavage And Polyadenylation

AbstractMost mammalian genes are subject to Alternative cleavage and PolyAdenylation (APA), often resulting in alternative length 3′ UTR isoforms. Thousands of extended or long 3′ UTR variants are preferentially expressed in neuron-enriched tissues of metazoans. However, the in vivo functions of these long 3′ UTR isoforms are largely unknown. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that is required for correct neural development. Calm1 generates short (Calm1-S) and long 3′ UTR (Calm1-L) mRNA isoforms via APA. We found Calm1-S to be broadly expressed across mouse tissues, whereas Calm1-L expression was largely restricted to neural tissues, including the dorsal root ganglion (DRG). Using CRISPR-Cas9 genome editing, a series of mouse deletion lines were generated that successfully eliminated expression of Calm1-L while maintaining expression of Calm1-S. One of these lines, Calm1Δ3′ UTR, carried a 163 bp deletion surrounding the distal polyA site. Examination of Calm1Δ3′ UTR embryos revealed disrupted development of the DRG. In Calm1Δ3′ UTR DRG explant cultures undergoing axon outgrowth, we observed a dramatic increase in axon fasciculation. These results demonstrate a physiological role for Calm1-L in DRG development, and more generally, establish a genome-editing strategy to study in vivo functions of long 3′ UTR isoforms.Author SummaryMore than half of all human genes generate alternative mRNA isoforms which differ in the length of their 3’ Untranslated regions (3’ UTRs). Through a process called Alternative Cleavage and Polyadenylation thousands of broadly expressed genes preferentially express long 3’ UTR variants in brain tissues whereas their short 3’ UTR counterparts are more broadly expressed. A challenge to study the functions of these transcripts has been to generate loss of function mutant animals that lack a long 3’ UTR isoform but maintain expression of the corresponding short 3’ UTR isoform. Here, we used the precise, rapid, and efficient approach of CRISPR genome-editing to generate long 3’ UTR mutant mice. These mice, which do not express the long 3’ UTR of the Calmodulin 1 (Calm1) gene, exhibit impairment in the development of sensory neurons, including increased fasciculation of axons and aberrant cell body migration. This finding is important because it provides conclusive genetic evidence for a neural function of a long 3’ UTR isoform in an animal. The CRISPR genome-editing approach used here can be applied to the study of neuron-enriched long 3’ UTR isoforms, which number in the thousands and have largely unexplored functions.

scholarly journals Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

2018 ◽  
Vol 115 (31) ◽  
pp. E7379-E7388 ◽  
Author(s):  
Laura J. Smith ◽  
Jason Wright ◽  
Gabriella Clark ◽  
Taihra Ul-Hasan ◽  
Xiangyang Jin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Homologous Recombination ◽  
Genome Editing ◽  
High Efficiency ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
A Genome ◽  
Component Approach ◽  
Il2rg Gene

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

scholarly journals Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1744
Author(s):  
Shingo Nakamura ◽  
Naoko Ando ◽  
Satoshi Watanabe ◽  
Eri Akasaka ◽  
Masayuki Ishihara ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Genome Editing ◽  
Fluorescent Protein ◽  
Guide Rna ◽  
Gene Insertion ◽  
Fetal Toxicity ◽  
A Genome ◽  
Wild Type Female ◽  
Green Fluorescent ◽  
Exogenous Plasmid

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

scholarly journals The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Benedetta M. Motta ◽  
Peter P. Pramstaller ◽  
Andrew A. Hicks ◽  
Alessandra Rossini
Keyword(s):  
Genome Editing ◽  
Ethical Issues ◽  
Therapeutic Potential ◽  
Gene Knockout ◽  
Model Systems ◽  
Large Deletions ◽  
Induced Pluripotent ◽  
The Impact

Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future.

scholarly journals Targeted editing and evolution of engineered ribosomes in vivo by filtered editing

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Felix Radford ◽  
Shane D. Elliott ◽  
Alanna Schepartz ◽  
Farren J. Isaacs
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
23S Rrna ◽  
Translational Efficiency ◽  
Rna Molecules ◽  
Genetic Elements ◽  
A Genome ◽  
Self Splicing

AbstractGenome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

scholarly journals Genome editing and ‘disenhancement’: Considerations on issues of non-identity and genetic pluralism

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Oliver Feeney ◽  
Vojin Rakić
Keyword(s):  
Genome Editing ◽  
Deaf Child ◽  
Genetic Selection ◽  
Deaf Children ◽  
Genetic Traits ◽  
Genomic Technologies ◽  
Procreative Beneficence ◽  
A Genome ◽  
Or Gene ◽  
The Relationship

AbstractIn the decade prior to CRISPR-Cas9, Michael Parker criticised Julian Savulescu’s Procreative Beneficence (PB) Principle by arguing against the confidence to know what’s best in terms of genetic traits for our offspring. One important outcome of this criticism was a greater moral acceptance of deaf people genetically selecting deaf children. Although this outcome may have been morally controversial in an impersonal harm context, in such genetic selection (PGD) cases, a deaf child is not harmed in person-affecting terms because no other life is available to that child. We highlight that the person-affecting versus impersonal harm distinction is still held by many as making a significant moral difference to their overall argument (i.e. Savulescu, Parker, Boardman, De Miguel Beriain) and so for the purposes of this paper, we will assume it makes ‘some difference’ (even if only at the level of the message it sends out). Insofar as one considers the presence person-affecting harm to be morally important (and to whatever extent), the impersonal harm context in which the Parker–Savulescu debate arose thereby blunts an arguably even more radical outcome—that of genetically engineering, or gene editing, deafness into pre-existing embryos of future children. Now, the potential of CRISPR-Cas9 has revitalised such debates by reframing impersonal and person-affecting benefits/harms in the context of such disputes on the harm or not of a (chosen) disability. Replacing the genetic selection context with a genome editing context, we argue that Parker’s argument should also deem it morally acceptable for people who are deaf to genetically edit embryos to become children who are also deaf. Felicity Boardman’s recent comments suggest a similar radical potential as Parker’s, with the radicalness also blunted by an impersonal context (a context that Boardman, at least, sees as significant). We conclude that the genome editing reframing will push such arguments beyond what were originally intended, and this will create a more radical message that may help further define the relationship between new genomic technologies and disability.

Multiple Thrombosis Modifier Loci Identified Using a Sensitized Mutagenesis Screen

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3755-3755
Author(s):  
Steven J Grzegorski ◽  
Yang Liu ◽  
Megan S Rost ◽  
Andy H Vo ◽  
Catherine Richter ◽  
...  
Keyword(s):  
Genome Editing ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Wild Type ◽  
High Fecundity ◽  
Mutagenesis Screen ◽  
Thrombin Activity ◽  
A Genome

Abstract Thrombosis is a leading cause of mortality in the developed world. Of the natural anticoagulants, congenital deficiencies of antithrombin III (AT3) lead to the highest risk of venous thromboembolism. However, the lifetime risk of thrombosis for these patients is only ~30%, presumably due to unknown modifier genes. Danio rerio (zebrafish) demonstrate a high degree of conservation of the coagulation cascade. Combined with their high fecundity, optically transparent embryos and well-characterized genetics, they offer an ideal model for low-cost high-throughput in vivo screens. Previously, we generated and described a knockout of at3 in zebrafish using genome editing. Homozygous mutant adults exhibit a decreased lifespan due to massive intracardiac thrombosis by 6 months of age, yet embryos survive spontaneous thrombosis with a consumptive coagulopathy characteristic of disseminated intravascular coagulation (DIC). This embryonic phenotype results in fibrinogen depletion and they are unable to form occlusive thrombi following laser-mediated venous endothelial injury. Here we use our at3-/- mutant line as a sensitized hypercoagulable background to identify novel modifiers of thrombosis. We performed a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen to discover dominant suppressor mutations that lead to increased survival. ENU treated at3+/- males were crossed with at3+/- females and offspring genotyped at 2-4 months of age. 53 at3-/- mutants survived beyond 7 months, 23 produced offspring, and 5 established permanent lines. One mutant, frost, also demonstrated reversal of embryonic DIC. Genomic DNAs from three frost mutants were evaluated by whole exome sequencing. An initial search of 54 known platelet and coagulation factor loci revealed a candidate missense mutation, C559F, in a highly conserved residue of the prothrombin (F2) heavy chain. The predicted deleterious mutation was not present in wild type stocks, zebrafish variant databases or the original mutagenized male. By 3 months of age, at3-/- juveniles heterozygous for the C559F allele demonstrated significantly improved survival compared to wild type siblings (p<0.0001). At 2 years of age, all at3-/- survivors were heterozygous for C559F (n=22). In order to definitively prove the f2 locus as the underlying cause of frost, we used TALEN genome editing to produce a separate knockout allele f2Δ, and bred that into the non-mutagenized at3-/- background. We found a significant increase in f2+/Δ;at3-/- survivors compared to f2+/+;at3-/- and f2Δ/Δ;at3-/- (p=0.006), demonstrating that partial knockdown of thrombin activity rescues at3-/- adult lethality, and confirming f2 as the locus underlying the frost mutation. Among f2+/+;at3-/- embryos 82% exhibit DIC, but this decreases to 51% in f2+/C559F;at3-/-. However, f2Δ does not have the same effect in the non-mutagenized at3-/- background with 85% of f2+/Δ;at3-/- embryos demonstrating DIC. This suggests that thrombin deficiency is necessary to unmask a phenotypic rescue due to a putative second modifier in the frost mutant background. Taken together, these data provide proof of principle for the validity of this screen and evidence that we have the power to identify novel dominant modifiers of prothrombin expression or thrombin function, including molecules outside of the canonical coagulation cascade. Furthermore, our data provide evidence for a second unknown locus modifying the embryonic DIC phenotype and acting epistatic to thrombin activity. Detection and characterization of this underlying gene may provide insight into the variable penetrance of thrombosis in patients with thrombophilic risk factors and offer new therapeutic options for treatment of DIC. Disclosures No relevant conflicts of interest to declare.

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