scholarly journals Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids

2019 ◽  
Author(s):  
Justin A. Bosch ◽  
Ryan Colbeth ◽  
Jonathan Zirin ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Germ Line ◽  
Insertion Site ◽  
End Joining ◽  
Endogenous Proteins ◽  
Non Homologous End Joining ◽  
Universal Donor

AbstractTargeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and non-homologous end joining (NHEJ) to insert universal donor plasmids into the genome. This method is a simple and fast alternative to HDR for certain strategies such as C-terminal tagging and gene disruption. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-RFP into seven genes in the fly germ line, producing heritable loss of function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, indels always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Finally, we apply this fast knock-in method to uncharacterized small open reading frame (smORF) genes. Therefore, homology-independent insertion is a useful genome editing technique in Drosophila that will better enable researchers to dissect gene function.Article summaryWe report a fast and simple genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. We demonstrate its usefulness in cultured cells to fluorescently tag endogenous proteins and in the fly germ line to generate heritable insertions that disrupt gene function and can act as expression reporters.

scholarly journals High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Haining Zhong ◽  
Cesar C Ceballos ◽  
Crystian I Massengill ◽  
Michael A Muniak ◽  
Lei Ma ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Gene Editing ◽  
Target Gene ◽  
Somatic Cells ◽  
High Fidelity ◽  
End Joining ◽  
Process Error ◽  
Endogenous Proteins ◽  
Non Homologous End Joining

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

scholarly journals High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

2020 ◽  
Author(s):  
Haining Zhong ◽  
Crystian I. Massengill ◽  
Michael A. Muniak ◽  
Lei Ma ◽  
Maozhen Qin ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Gene Editing ◽  
Target Gene ◽  
Somatic Cells ◽  
High Fidelity ◽  
End Joining ◽  
Process Error ◽  
Endogenous Proteins ◽  
Non Homologous End Joining

ABSTRACTPrecise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

scholarly journals Precise genome engineering inDrosophilausing prime editing

2020 ◽  
Vol 118 (1) ◽  
pp. e2021996118
Author(s):  
Justin A. Bosch ◽  
Gabriel Birchak ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Germ Line ◽  
Point Mutations ◽  
Model Organism ◽  
Model Organisms ◽  
Marker Genes ◽  
Germ Line Transmission

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organismDrosophila melanogasterand developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes,ebony,white, andforked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit inebonyto 36% of progeny. Our results suggest that prime editing is a useful system inDrosophilato study gene function, such as engineering precise point mutations, deletions, or epitope tags.

Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of non-homologous end joining using CRISPR/Cas9

2021 ◽  
Author(s):  
Jun-Liang Tu ◽  
Xin-Yuan Bai ◽  
Yong-Liang Xu ◽  
Na Li ◽  
Jun-Wei Xu
Keyword(s):  
Homologous Recombination ◽  
Functional Genomics ◽  
Ganoderma Lucidum ◽  
Gene Disruption ◽  
Target Gene ◽  
End Joining ◽  
Gene Insertion ◽  
Replacement Method ◽  
Low Efficiency ◽  
Non Homologous End Joining

Targeted gene insertion or replacement is a promising genome editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum , targeted gene insertion and replacement remains a serious challenge due to the low efficiency of homologous recombination (HR) in these species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the non-homologous end joining pathway (NHEJ) at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma , we first inactivated the NHEJ pathway via disruption of the Ku70 gene ( ku70 ) using a dual sgRNA-directed gene deletion method. Disruption of the ku70 significantly decreased NHEJ activity in G. lucidum . Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of a targeted gene insertion and replacement when target DNA orotidine 5’-monophosphate decarboxylase gene ( ura3 ) with 1.5 kb 5’ and 3’ homologous flanking sequences were used as a donor template, compared to 3.3% and 0% for a control strain (Cas9 strain) at these targeted sites, respectively. Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum . Importance Functional genomic studies have been hindered in Ganoderma by the absence of adequate genome engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum , targeted gene insertion and replacement has remained a serious challenge due to the low efficiency of homologous recombination in these species, although such precise genome modifications including site mutations, site-specific integrations and allele or promoter replacements would be incredibly valuable. In this work, we inactivated the non-homologous end joining repair mechanism in G. lucidum by disrupting the ku70 using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70 -disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

scholarly journals Characterization of Sleeping Beauty Transposition and Its Application to Genetic Screening in Mice

2003 ◽  
Vol 23 (24) ◽  
pp. 9189-9207 ◽  
Author(s):  
Kyoji Horie ◽  
Kosuke Yusa ◽  
Kojiro Yae ◽  
Junko Odajima ◽  
Sylvia E. J. Fischer ◽  
...  
Keyword(s):  
Large Scale ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Donor Site ◽  
Insertion Site ◽  
Sleeping Beauty ◽  
Mutant Mice ◽  
Genome Wide ◽  
Green Fluorescent

ABSTRACT The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.

scholarly journals Precise genome engineering in Drosophila using prime editing

2020 ◽  
Author(s):  
Justin A. Bosch ◽  
Gabriel Birchak ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Germ Line ◽  
Point Mutations ◽  
Model Organism ◽  
Model Organisms ◽  
Marker Genes ◽  
Germ Line Transmission

AbstractPrecise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely installed premature stop codons in three classical visible marker genes, ebony, white, and forked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ line transmission of a precise edit in ebony to ~50% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.

scholarly journals In Vivo Blunt-End Cloning Through CRISPR/Cas9-Facilitated Non-Homologous End-Joining

2015 ◽  
Author(s):  
Jonathan M Geisinger ◽  
Sören Turan ◽  
Sophia Hernandez ◽  
Laura P Spector ◽  
Michele P Calos
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
Fluorescent Protein ◽  
Double Strand Breaks ◽  
Hek293 Cells ◽  
End Joining ◽  
Independent Manner ◽  
Strand Breaks ◽  
Non Homologous End Joining

The ability to precisely modify the genome in a site-specific manner is extremely useful. The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting this excision and repair to insert exogenous sequences in a homology-independent manner without loss of additional nucleotides. We successfully utilize this method in a human immortalized cell line and induced pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P destabilization domain for investigating repair dynamics of Cas9-induced double-strand breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

scholarly journals Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity

2021 ◽  
Author(s):  
Anie Day D C Asa ◽  
Rujira Wanotayan ◽  
Mukesh Kumar Sharma ◽  
Kaima Tsukada ◽  
Mikio Shimada ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Strand Break ◽  
Growth Defect ◽  
Wild Type ◽  
End Joining ◽  
Dsb Repair ◽  
Stable Transfectants ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Green Fluorescent

Abstract Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.

scholarly journals Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression

2017 ◽  
Author(s):  
Yuping Huang ◽  
Caitlin McCann ◽  
Andrey Samsonov ◽  
Dmitry Malkov ◽  
Gregory D Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Cell Cycle Control ◽  
Strand Break ◽  
End Joining ◽  
Genetic Changes ◽  
A Cell ◽  
Non Homologous End Joining ◽  
Green Fluorescent ◽  
Chromosomal Sequences

ABSTRACTTargeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integration of donor ssDNA. Most importantly, the ratio of ssDNA donor integration to non-homologous end joining (NHEJ) was observed to increase, suggesting that cell cycle control Cas9 expression may be an effective strategy to bias DNA repair outcomes.

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