Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells

Cromwell, Christopher R; Jovel, Juan; Hubbard, Basil P.

doi:10.1007/978-1-0716-0687-2_11

Cited by 4 publications

(7 citation statements)

References 28 publications

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“…293T cells stably expressing Cas9 were transfected using lipofectamine RNAiMAX with sgRNAs according to the manufacturer’s instructions to a final concentration of 30 nM. , …”

Section: Methodsmentioning

confidence: 99%

“…293T cells stably expressing Cas9 were transfected using lipofectamine RNAiMAX with sgRNAs according to the manufacturer's instructions to a final concentration of 30 nM. 8,9 Cellular Cleavage Assays. Cellular cleavage assays were performed as previously described.…”

Section: Bioconjugate Chemistrymentioning

confidence: 99%

“…CRISPR-Cas9 has enabled gene editing both in the lab, − allowing for the function of proteins to be readily interrogated, − and in the clinic, where it promises to offer treatments for diseases such as sickle cell anemia and cystic fibrosis. − Cas9 directs sequence-specific DNA cleavage through the aid of a guide RNA (gRNA) consisting of a CRISPR RNA (crRNA), which contains a 20 nucleotide (nt) programmable target sequence and a trans-activating crRNA (tracrRNA) that binds Cas9. The tracrRNA acts as a bridge between crRNA and the enzyme .…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs

et al. 2022

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Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5′-azide and 3′-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne–azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

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“…293T cells stably expressing Cas9 were transfected using lipofectamine RNAiMAX with sgRNAs according to the manufacturer’s instructions to a final concentration of 30 nM. , …”

Section: Methodsmentioning

confidence: 99%

Section: Bioconjugate Chemistrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cells stably expressing Cas9 were transfected using Lipofectamine RNAiMAX with sgRNAs according to the manufacturer's instructions to a final concentration of 30 nM. 8,9 Cellular cleavage assays. 48 hours after transfection, genomic DNA (gDNA) was isolated from transfected cells using the DNeasy Blood & Tissue Kit (Qiagen) and quantified using a NanoPhotometer NP80 (Implen).…”

Section: Methodsmentioning

confidence: 99%

“…CRISPR-Cas9 has enabled gene editing both in the lab, [1][2][3][4][5][6][7][8][9] allowing for the function of proteins to be readily interrogated, [10][11][12] and in the clinic, where it promises to offer treatments for diseases such as sickle cell anemia and cystic fibrosis. [13][14][15][16][17][18] Cas9 directs sequence specific DNA cleavage through the aid of a CRISPR RNA (crRNA), which contains a 20-nucleotide (nt) programmable target sequence, and a trans-activating crRNA (tracrRNA) that binds the Cas9.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Click Enables Multi-Gene Editing with Modular Synthetic sgRNAs

Park

Osman

Cromwell

et al. 2021

Preprint

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Gene editing systems such as CRISPR/Cas9 readily enable individual gene phenotypes to be studied through loss-of-function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR/Cas9 to disrupt individual loci, technical limitations in single guide RNA (sgRNA) synthesis hinder the expansion of this approach to multi-color cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-assisted cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 3’-azide and 5’-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition (CuAAC) to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNA in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating doubly-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

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