An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

Kanca, Oguz; Zirin, Jonathan; García-Marqués, Jorge; Knight, Shannon M.; Yang‐Zhou, Donghui; Amador, Gabriel; Chung, Hyunglok; Zuo, Zhuang; Ma, Liwen; He, Yuchun; Lin, Wen–Jen; Fang, Ying; Ge, Ming; Yamamoto, Shinya; Schulze, Karen L.; Hu, Yanhui; Spradling, Allan C.; Mohr, Stephanie E.; Perrimon, Norbert; Bellen, Hugo J.

doi:10.7554/elife.51539

Cited by 132 publications

(121 citation statements)

References 67 publications

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“…Current Protocols in Molecular Biology of several Drosophila cell lines can be queried based on modENCODE Drosophila cell line transcriptomics data sets (Cherbas et al, 2011), for example, using the Drosophila Gene Expression Tool (DGET;https://www.flyrnai.org/dget;Hu, Comjean, Perrimon, & Mohr, 2017). We note that Kanca et al (2019) report isolation of GFP-tagged cell lines using the ssDNA Drop-In approach for some targets expressed at moderate or low levels.…”

Section: Of 28mentioning

confidence: 99%

“…This protocol describes a method for CRISPR-mediated knock-in of a fluorescent protein that relies on an ssDNA donor to provide the fluorescent protein ORF as an artificial exon, referred to as the ssDNA Drop-In method (Kanca et al, 2019). Following design of the knock-in and corresponding ssDNA and sgRNA, these molecular reagents are generated and transfected into cells.…”

Section: Knock-in Into Cas9-positive S2r+ Cells Using the Ssdna Drop-mentioning

confidence: 99%

“…The efficiency of introduction of tags into Drosophila cells is dramatically improved by introduction of the CRISPR-Cas9 system as a tool to facilitate insertion or 'knock in' of an insertion cassette into a specific locus. Indeed, CRISPR approaches have been successfully used to generate Drosophila cells in which endogenous loci are tagged by GFP fusion, e.g., Bosch, Colbeth, Zirin, & Perrimon, 2019;Bottcher et al, 2014;Kanca et al, 2019;Kunzelmann, Bottcher, Schmidts, & Forstemann, 2016;Wang et al, 2016. The standard protocol involves production of a plasmid donor with ß500to 1000bp homology arms (Housden & Perrimon, 2016). Alternative approaches, as presented here, can accelerate the CRISPR knock-in workflow, for example by making it easier to obtain or prepare donor constructs (Bosch et al, 2019;Kanca et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Indeed, CRISPR approaches have been successfully used to generate Drosophila cells in which endogenous loci are tagged by GFP fusion, e.g., Bosch, Colbeth, Zirin, & Perrimon, 2019;Bottcher et al, 2014;Kanca et al, 2019;Kunzelmann, Bottcher, Schmidts, & Forstemann, 2016;Wang et al, 2016. The standard protocol involves production of a plasmid donor with ß500to 1000bp homology arms (Housden & Perrimon, 2016). Alternative approaches, as presented here, can accelerate the CRISPR knock-in workflow, for example by making it easier to obtain or prepare donor constructs (Bosch et al, 2019;Kanca et al, 2019). Specifically, we present, as alternatives to the standard approach, a single-stranded DNA (ssDNA) "Drop-In" method based on in vitro synthesis of an ssDNA donor (Kanca et al, 2019; Basic Protocol 1) and a "CRISPaint"-based approach that relies on universal donors…”

Section: Introductionmentioning

confidence: 99%

“…Alternative approaches, as presented here, can accelerate the CRISPR knock-in workflow, for example by making it easier to obtain or prepare donor constructs (Bosch et al, 2019;Kanca et al, 2019). Specifically, we present, as alternatives to the standard approach, a single-stranded DNA (ssDNA) "Drop-In" method based on in vitro synthesis of an ssDNA donor (Kanca et al, 2019; Basic Protocol 1) and a "CRISPaint"-based approach that relies on universal donors…”

Section: Introductionmentioning

confidence: 99%

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Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

Bosch

Knight

Kanca

et al. 2019

CP Molecular Biology

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The CRISPR‐Cas9 system makes it possible to cause double‐strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or ‘knock‐in’ of an exogenous cassette. One common application of knock‐in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock‐in of fluorescent protein ORFs into Cas9‐expressing Drosophila S2R+ cultured cells, the single‐stranded DNA (ssDNA) Drop‐In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop‐In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock‐in into Cas9‐positive S2R+ cells using the ssDNA Drop‐In approach Basic Protocol 2: Knock‐in into Cas9‐positive S2R+ cells by homology‐independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+‐MT::Cas9 Drosophila cells Support Protocol 5: Single‐cell isolation of fluorescent cells using FACS

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Section: Of 28mentioning

confidence: 99%

Section: Knock-in Into Cas9-positive S2r+ Cells Using the Ssdna Drop-mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

Bosch

Knight

Kanca

et al. 2019

CP Molecular Biology

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CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

et al. 2021

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Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of CRISPR-Cas9 technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate or knockout specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knockin strategies of genes in typical diploid cells (i.e. cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein, we detail our methodology for replacing endogenous proteins with intended knockin mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele-frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling,

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Single‐strand annealing: Molecular mechanisms and potential applications in CRISPR‐Cas‐based precision genome editing

Das

Nguyen

et al. 2021

Biotechnology Journal

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Background: Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival.Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown.Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. Purpose and Scope:Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. Summary and Conclusion:The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5′-3′ resection to reveal homologous repeats on the 3′ ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.

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An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

Cited by 132 publications

References 67 publications

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

Single‐strand annealing: Molecular mechanisms and potential applications in CRISPR‐Cas‐based precision genome editing

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