Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems

Filippova, Julia A.; Matveeva, Anastasiya M.; Zhuravlev, Evgenii; Степанов, Г. А.

doi:10.1016/j.biochi.2019.09.003

Cited by 65 publications

(43 citation statements)

References 84 publications

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“…The single gRNA more commonly used in tool applications is a fusion of the tracrRNA and crRNA through a short RNA loop between the repeat-anti-repeat sequences in the Upper Stem (Figure 1 ) ( 4 ). An important area of research has been the manipulation of the gRNA sequence and structure, through introduction of extra or modified nucleotides, and/or the addition of folded RNA aptamers ( 7 , 8 ). Using a combination of ensemble and single-molecule biochemical assays, we examined how gRNA modification affects ribonucleoprotein (RNP) assembly, R-loop stability and DNA cleavage.…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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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“…The specificity of this system is conferred by the interaction of the Cas9 nuclease with an RNA composed of a scaffold RNA joined to a guide RNA (gRNA), which directs the protein to a specific target DNA sequence for cleavage [4]. The site of mutagenesis can therefore be programmed simply by adjusting the sequence of the gRNA, provided a protospacer adjacent motif (PAM) sequence is present in the target DNA [4,5]. The CRISPR/Cas9 system is also useful for other genetic manipulations beyond genomic sequence editing, such as regulation of gene expression and epigenetic modification [2,4].…”

Section: Introductionmentioning

confidence: 99%

A CRISPR/dCas9 toolkit for functional analysis of maize genes

et al. 2020

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Background The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

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“…The speci city of this system is conferred by the interaction of the Cas9 nuclease with an RNA composed of a scaffold RNA joined to a guide RNA (gRNA), which directs the protein to a speci c target DNA sequence for cleavage [4]. The site of mutagenesis can therefore be programmed simply by adjusting the sequence of the gRNA, provided a protospacer adjacent motif (PAM) sequence is present in the target DNA [4,5]. The CRISPR/Cas9 system is also useful for other genetic manipulations beyond genomic sequence editing, such as regulation of gene expression and epigenetic modi cation [2,4].…”

Section: Introductionmentioning

confidence: 99%

A CRISPR/dCas9 toolkit for functional analysis of maize genes

Gentzel

Park

Bellizzi

et al. 2020

Preprint

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Background: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

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Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems

Cited by 65 publications

References 84 publications

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

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

A CRISPR/dCas9 toolkit for functional analysis of maize genes

A CRISPR/dCas9 toolkit for functional analysis of maize genes

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