Versatile 3′ Functionalization of CRISPR Single Guide RNA

Palumbo, Cody M.; Gutierrez‐Bujari, Jeton M.; O’Geen, Henriette; Segal, David J.; Beal, Peter A.

doi:10.1002/cbic.201900736

Cited by 12 publications

(13 citation statements)

References 47 publications

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“…Consistent with this, these gRNA features have been shown to accommodate RNA aptamer scaffolds with a range of sequences and sizes, e.g. ( 19 , 34–37 ). Often gRNA fusions are used to provide a binding sequence to colocalise a protein or fluorescent label and are hence used with dCas9 rather than nuclease active WT Cas9.…”

Section: Discussionmentioning

confidence: 58%

5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

Mullally

Aelst

Naqvi

et al. 2020

Nucleic Acids Research

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Abstract A key aim in exploiting CRISPR–Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.

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Section: Discussionmentioning

confidence: 58%

5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

Mullally

Aelst

Naqvi

et al. 2020

Nucleic Acids Research

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“…28 While these approaches use DNA-encoded strategies, the direct introduction of a gRNA to cells has been shown to have advantages. 6,17,[29][30][31][32][33][34][35][36][37] For example, direct introduction of synthetic crRNA and tracrRNA made by solid-phase, rather than enzyme-catalyzed synthesis, allows unnatural chemical modifications to be incorporated that confer benefits, such as increased stability, [31][32][33] reduced off-target effects, 34,35 and fluorescent labeling. [35][36][37] Accordingly, an attractive approach to multiplexing genome editing that alleviates the need for in-cell processing is to introduce multiple gRNAs simultaneously into a cell.…”

Section: Introductionmentioning

confidence: 99%

“…6,17,[29][30][31][32][33][34][35][36][37] For example, direct introduction of synthetic crRNA and tracrRNA made by solid-phase, rather than enzyme-catalyzed synthesis, allows unnatural chemical modifications to be incorporated that confer benefits, such as increased stability, [31][32][33] reduced off-target effects, 34,35 and fluorescent labeling. [35][36][37] Accordingly, an attractive approach to multiplexing genome editing that alleviates the need for in-cell processing is to introduce multiple gRNAs simultaneously into a cell. This can be achieved by complexing multiple guides using a nanoparticle delivery agent, 38,39 or introducing multiple gRNAs targeting different genes into the same cell.…”

Section: Introductionmentioning

confidence: 99%

“…This inability to produce, purify, and characterize sizable amounts of synthetic sgRNA also limits the generation of welldefined hybrid materials that facilitate cellular delivery of multiple gene-editing constructs. 52 One strategy that has been explored to increase the amount and structural diversity of sgRNA involves synthesizing it from two smaller fragments using enzyme-catalyzed 37,53 or bioconjugation techniques. 50,51 The first reported example of the latter involved the introduction of a hexyne phosphoramidite monomer at the 5' end of the tracrRNA and a 3'-amine modification on the crRNA further functionalized with azido-acetic acid NHS ester.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…Palumbo et al recently designed sgRNA bearing a variety of succinyl esters at the 3′ end including an iodoacetamide functional handle, strained cyclooctyne, Cy5 fluorophore, and biotin which had negligible effects on cleavage efficiency. 445 Using the iodoacetamide functional handle they were able to achieve crosslinking with a mutant Cas9 protein and the biotinylated handle allowed for identification of three human proteins that bind to sgRNA. Wang et al recently blocked important 2′OH contact points between sgRNA and the Cas9 protein with photolabile groups, thereby preventing formation of the active complex.…”

mentioning

confidence: 99%