sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing

Graf, Robin; Li, Xun; Chu, Van Trung; Rajewsky, Klaus

doi:10.1016/j.celrep.2019.01.024

Cited by 101 publications

(117 citation statements)

References 18 publications

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“…The sgRNA simplifies the assembly of the ribonuclear protein (RNP) complex as well as the transcription of the gRNA from a delivered plasmid. Essential parts of the sgRNA have been investigated and many studies have been carried out to optimize the sgRNA sequence . However, for therapeutic applications of the CRISPR/Cas9 system that involve delivery of an intact RNP, chemical modifications of the sgRNA will be required, similar to other existing therapeutic oligonucleotides (i.e., antisense oligonucleotides, siRNAs) .…”

Section: Introductionmentioning

confidence: 99%

Versatile 3′ Functionalization of CRISPR Single Guide RNA

et al. 2020

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Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3′‐end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3′‐end was broadly tolerated by Streptococcus pyogenes Cas9 in an in vitro DNA cleavage assay. The 3′‐biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA‐binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.

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Section: Introductionmentioning

confidence: 99%

Versatile 3′ Functionalization of CRISPR Single Guide RNA

et al. 2020

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“…The observation that no editing activity was detected at the StGBSSI target locus for both spacer lengths (sgRNA1 and 3) may be due to the presence of an inefficient motif in the spacer sequence. However, none of the two motifs identified as inefficient in a previous study [20] was present in our spacer sequences, suggesting another origin for the lack of editing at this locus, such as the genomic context that may interfere with Cas9 binding and cleavage, and pointing out to the necessity to test independent spacer sequences for a target gene in order to maximize the likelihood of successful editing. Based on manual analysis of the chromatograms for sgRNA2 and 4 (StDMR6-1 locus), we found that frameshift mutations mostly occurred about 4-bp upstream of the PAM sequence ( Fig 2B), as previously reported for SaCas9 [11,12].…”

Section: Crispr-sacas9-mediated Gene Editing Of the Potato Genomementioning

confidence: 60%

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato

Veillet

Kermarrec

Chauvin

et al. 2020

Preprint

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Genome editing is now widely used in plant science for both fundamental research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows to edit many sites in plant genomes. This system has been highly successful to produce of knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on request nucleotide conversion, allowing to fine-tune protein function and generate gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to simultaneously mediate different catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing to precisely modify specific residues or regulatory elements in potato. Our proof-of-concept results in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.

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“…For our simulations, we used the decile of GC content for each sgRNA's template guide. Users may also derive a sgRNA categorical feature through an efficiency score formula such as those developed in (24,(31)(32)(33)(34)(35). Guide efficiency g is derived from fitting weight ω f to the feature category F g of guide g:…”

Section: Methodsmentioning

confidence: 99%

ACE: A Probabilistic Model for Characterizing Gene-Level Essentiality in CRISPR Screens

Hutton

Siepel

2019

Preprint

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High-throughput knockout screens based on CRISPR-Cas9 are widely used to evaluate the essentiality of genes across a range of cell types. Here we introduce a probabilistic modeling framework, Analysis of CRISPR-based Essentiality (ACE), that enables new statistical tests for essentiality based on the raw sequence read counts from such screens. ACE estimates the essentiality of each gene using a flexible likelihood framework that accounts for multiple sources of variation in the CRISPR-Cas9 experimental process. In addition, the method can identify genes that differ in their degree of essentiality across samples using a likelihood ratio test. We show using simulations that ACE is competitive with the best available methods in predicting essentiality, and is especially useful for the identification of differential essentiality. Furthermore, by applying ACE to publicly available CRISPR-screen data, we are able to identify both known and previously overlooked candidates for genotype-specific essentiality, including RNA m6-A methyltransferases that exhibit enhanced essentiality in the presence of inactivating TP53 mutations. In summary, ACE provides improved quantification of essentiality specific to cancer subtypes, and a robust probabilistic framework for identifying genes responsive to therapeutic targeting.

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sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing

Cited by 101 publications

References 18 publications

Versatile 3′ Functionalization of CRISPR Single Guide RNA

Versatile 3′ Functionalization of CRISPR Single Guide RNA

CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato

ACE: A Probabilistic Model for Characterizing Gene-Level Essentiality in CRISPR Screens

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