scholarly journals Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

2017 ◽  
Author(s):  
Eric J. Aird ◽  
Klaus N. Lovendahl ◽  
Amber St. Martin ◽  
Reuben S. Harris ◽  
Wendy R. Gordon
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Repair Efficiency ◽  
Rnp Complex ◽  
Multiple Cell ◽  
Low Efficiency ◽  
A Site ◽  
Protein Tags

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome where the Cas9 nuclease then induces a double stranded break (DSB)1,2. The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB3,4. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded donor oligonucleotide (ssODN) to the Cas9/guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease5, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to an 8-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci, and is more pronounced at low RNP concentrations.

scholarly journals Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

2021 ◽  
Author(s):  
Xiangfeng Kong ◽  
Zikang Wang ◽  
Yingsi Zhou ◽  
Xing Wang ◽  
Linyu Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Coding Rna ◽  
Bacterial Genetic ◽  
Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

scholarly journals CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

2015 ◽  
Vol 1 (7) ◽  
pp. e1500454 ◽  
Author(s):  
Xin Han ◽  
Zongbin Liu ◽  
Myeong chan Jo ◽  
Kai Zhang ◽  
Ying Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Function Analysis ◽  
Embryonic Stem ◽  
Cell Types ◽  
Disease Genes ◽  
Deformation Method ◽  
Targeting Therapy ◽  
Membrane Deformation ◽  
Guide Rna ◽  
Different Cell Types

The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.

scholarly journals Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

2019 ◽  
Vol 116 (42) ◽  
pp. 20959-20968 ◽  
Author(s):  
Sundaram Acharya ◽  
Arpit Mishra ◽  
Deepanjan Paul ◽  
Asgar Hussain Ansari ◽  
Mohd. Azhar ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Disorders ◽  
High Specificity ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Multiple Loci ◽  
Specificity Of Binding ◽  
Induced Pluripotent ◽  
Very High

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

scholarly journals Enhancement of homology-directed repair with chromatin donor templates in cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Grisel Cruz-Becerra ◽  
James T Kadonaga
Keyword(s):  
Genome Editing ◽  
Dna Fragments ◽  
Natural Form ◽  
Naked Dna ◽  
Homology Directed Repair ◽  
Coding Regions ◽  
Wide Range ◽  
Low Efficiency ◽  
Donor Template ◽  
In Cells

A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes.

scholarly journals Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals Efficient Homology-directed Repair with Circular ssDNA Donors

2019 ◽  
Author(s):  
Sukanya Iyer ◽  
Aamir Mir ◽  
Joel Vega-Badillo ◽  
Benjamin P. Roscoe ◽  
Raed Ibraheim ◽  
...  
Keyword(s):  
Genome Editing ◽  
K562 Cells ◽  
Cost Effective ◽  
Cell Types ◽  
Traffic Light ◽  
Homology Directed Repair ◽  
Mammalian Cell Lines ◽  
Cost Effective Method ◽  
Circular Ssdna ◽  
Fluorescent Tag

AbstractWhile genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

Use of Crispr/Cas Genome Editing in Ba/F3 Cells to Generate the Fip1l1-Pdgfra and Nup214-Abl1 Fusion Genes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3575-3575
Author(s):  
Marlies Vanden Bempt ◽  
Charles E de Bock ◽  
Nicole Mentens ◽  
Olga Gielen ◽  
Ellen Geerdens ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Expression ◽  
Genome Editing ◽  
Fusion Gene ◽  
Stop Codon ◽  
Chromosomal Rearrangements ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Fusion Protein Expression ◽  
A Cell

Abstract CRISPR/Cas genome editing is a powerful tool to precisely induce chromosomal breaks and to modify genes of interest. Cas9, an RNA-guided DNA endonuclease derived from Streptococcus pyogenes, is able to generate double stranded breaks (DSBs) in the genomic locus to where it is directed by its guide RNA (gRNA) component. The DSBs are subsequently repaired by one of the two main host repair mechanisms: the error-prone Non-homologous end joining (NHEJ) pathway or the very specific Homology-directed repair (HDR) pathway. We aimed to use CRISPR/Cas genome editing to generate the Fip1l1-Pdgfra and Nup214-Abl1 fusion genes by inducing chromosomal rearrangements in the interleukin-3 dependent Ba/F3 cell line. Prior to generating the chromosomal rearrangements, we optimized CRISPR/Cas genome editing in Ba/F3 cells, by targeting Cas9 to exon 24 of CD45, a cell surface transmembrane protein, of which inactivation can be easily detected by flow cytometry. Electroporation of Ba/F3 cells with plasmids expressing Cas9 and the specific guide RNA led to efficient inactivation of the CD45 gene, as measured by flow cytometry (30% of the cells showed loss of CD45 expression). The use of the Cas9 nickase variant led to an increased efficiency of CD45 inactivation with 58% of the cells showing loss of CD45 expression. We then extended these studies to assess the efficiency of homology-directed repair to introduce a specific mutation, using a single strand donor template to generate a premature stop codon in exon 24 of CD45. The successful introduction of the novel stop codon in CD45 was confirmed by PCR amplification of the targeted exon followed by massive parallel sequencing (MiSeq, Illumina) and we observed this endogenous mutation in 80% of the Ba/F3 clones. Having optimised the use and efficiency of CRISPR/Cas in Ba/F3 cells, we aimed to introduce double stranded breaks simultaneously in the genes Fip1l1 and Pdgfra to generate a cell based model for the FIP1L1-PDGFRA fusion gene as observed in chronic eosinophilic leukemia. Double strand breaks were introduced in Fip1l1 exon 23, 31, 32 or 34 together with simultaneous breaks in Pdgfra exon 12, both located on mouse chromosome 5. Upon IL3 removal, cells harbouring the deletion and fusion gene were able to survive, grow and form colonies in semi-solid medium, as was shown before for Ba/F3 cells transduced with retroviral vectors expressing FIP1L1-PDGFRA. The presence of the deletion was confirmed by PCR, and fusion protein expression was detected by Western blotting. A fusion between exon 1 of Fip1l1 and exon 12 of Pdgfra could also transform the cells, which confirmed earlier findings that the transforming capacities of the fusion protein are independent of Fip1l1 and dependent on the interruption of the juxtamembrane region of PDGFRA. The expression and phosphorylation levels of Fip1l1-Pdgfra were compared between the CRISPR/Cas generated Ba/F3 cells and retrovirally transduced cells overexpressing FIP1L1-PDGFRA. As expected, retrovirally transduced cells showed a much higher protein expression level of FIP1L1-PDGFRA, and much stronger phosphorylation compared to the CRISPR/Cas generated cells, in which the endogenous Fip1l1 promoter is used to drive the expression of the fusion protein. We also observed a difference in sensitivity to inhibition by imatinib, a kinase inhibitor with strong activity against PDGFRA. The same strategy was followed to generate a fusion between Nup214 and Abl1, as observed in a subset of T-cell acute lymphoblastic leukemia cases. Ba/F3 cells harbouring the Nup214-Abl1 fusion gene were able to survive and grow independent of IL3. The presence of the fusion gene was confirmed by PCR, and fusion protein expression was detected by Western blotting. Taken together, these data show that CRISPR/Cas induced chromosomal translocations in cells more faithfully recapitulate gene expression levels and sensitivity to chemotherapeutics when compared to retroviral transduction based expression of an oncogene. In conclusion, we have now designed and implemented an optimised platform to use CRISPR/Cas genome editing in Ba/F3 cells and measure gRNA efficacy by massive parallel sequencing. Our data confirm that the CRISPR/Cas genome editing system can be used to generate chromosomal rearrangements in Ba/F3 cells and provides a method to generate improved cell based models for the study of oncogenic tyrosine kinases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

2020 ◽  
Author(s):  
Haseena Khan ◽  
Megan C McDonald ◽  
Simon J Willams ◽  
Peter Solomon
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Pathogenic Fungus ◽  
Point Mutations ◽  
Dna Breaks ◽  
Gene Manipulation ◽  
Homology Directed Repair ◽  
Rnp Complex ◽  
Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (gRNA) which interact to form a Cas9-gRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and gRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting gene transgene-free knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

scholarly journals TedSim: temporal d​ynamics sim​ulation of single cell RNA-sequencing data and cell division history

2021 ◽  
Author(s):  
Xinhai Pan ◽  
Hechen Li ◽  
Xiuwei Zhang
Keyword(s):  
Cell Division ◽  
Single Cell ◽  
Genome Editing ◽  
Computational Methods ◽  
Temporal Dynamics ◽  
Cell Types ◽  
Sequencing Data ◽  
Gene Expressions ◽  
Cell Lineages ◽  
Multiple Cell

Recently, the combined scRNA-seq and CRISPR/Cas9 genome editing technologies have enabled simultaneous readouts of gene expressions and lineage barcodes, which allows for the reconstruction of the cell division tree, and makes it possible to trace the origin of each cell type. Computational methods are emerging to take advantage of the jointly profiled scRNA-seq and lineage barcode data to better reconstruct the cell division history or to infer the cell state trajectories. Here, we present TedSim (single cell Temporal dynamics Simulator), a simulator that simulates the cell division events from the root cell to present-day cells, simultaneously generating the CRISPR/Cas9 genome editing lineage barcodes and scRNA-seq data. In particular, TedSim generates cells from multiple cell types through cell division events. TedSim can be used to benchmark and investigate computational methods which use either or both of the two types of data, scRNA-seq and lineage barcodes, to study cell lineages or trajectories. TedSim is available at: https://github.com/Galaxeee/TedSim.

scholarly journals High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing

2018 ◽  
Vol 115 (42) ◽  
pp. E9944-E9952 ◽  
Author(s):  
Cory D. Sago ◽  
Melissa P. Lokugamage ◽  
Kalina Paunovska ◽  
Daryll A. Vanover ◽  
Christopher M. Monaco ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
High Throughput ◽  
Cell Types ◽  
Functional Protein ◽  
Guide Rna ◽  
High Throughput Method ◽  
Multiple Cell ◽  
In Vivo Screen ◽  
Cas9 Mrna

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.

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