Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

Brinkman, Eva K.; Chen, Tao; Haas, Marcel de; Holland, Hanna A.; Akhtar, Waseem; Steensel, Bas van

doi:10.1016/j.molcel.2018.04.016

Cited by 222 publications

(280 citation statements)

References 59 publications

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“…A crude estimate from Kim et al 29 suggested that indel formation by CRISPR/Cas occurs early in the process but is detected only after about fifteen hours. These data align with the belief that sequence specific nucleases direct indel formation only after multiple cycles of breakage and perfect repair 11 . Our data suggest that the time delay between penetration of the nucleus and detectable gene editing activity centers around four to eight hours, an observation that aligns with those made by Reid et al 30 .…”

Section: Discussionsupporting

confidence: 84%

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Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

Banas

Rivera-Torres²,

Bialk³

et al. 2019

Preprint

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Recent data suggest that a time lag exists between nuclear penetration and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed gene editing activity in human cells. As CRISPR/Cas approaches clinical implementation, it is critical to establish a biological time frame in which the complex enters the cell and nucleus and executes its gene editing function. We are developing CRISPR-directed gene editing for the treatment of non-small cell lung carcinoma focusing Nuclear Factor Erythroid 2-Related Factor-Like (NRF2), a transcription factor which regulates chemoresistance. In this report, we define cellular events that surround the initialization of CRISPR-directed gene editing as a function of time. We analyze the efficiency of cellular transfection of both components of the RNP particle and assess the emergence of indels. For the first time, we image the nuclear positioning of the tracrRNA and Cas9 as a complex and as individual gene editing components. Our results indicate that while the nuclear localization of the CRISPR/Cas complex is efficient and rapid, disruption of the NRF2 gene appears four to eight hours later. We reveal an initial snapshot of the schedule and processing of CRISPR/Cas in a lung cancer cell; information that will be useful as cell-based protocols are designed and advanced.

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Section: Discussionsupporting

confidence: 84%

“…Recent data suggest that a time lag exists between nuclear penetration and the appearance of gene editing activity in human cells 11 . For clinical implementation, it is essential that a biological time frame in which the complex enters the cell, penetrates the nuclease and executes its gene editing function is established.…”

Section: Introductionmentioning

confidence: 99%

Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

Banas

Rivera-Torres²,

Bialk³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…If a suitable template is provided, the cell can use homology-directed repair to integrate a novel allele or transgene at the targeted site (Ceasar, Rajan, Prykhozhij, Berman, & Ignacimuthu, 2016). Alternately, the cell can repair the lesion via nonhomologous end joining (NHEJ), an error-prone process that commonly results in an insertion or deletion (indel) mutation at the DSB location (Brinkman et al, 2018). In this way, CRISPR can be used to introduce stable, nonrevertible alterations to mammalian genes.…”

Section: Introductionmentioning

confidence: 99%

Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Giuliano

Lin

Girish

et al. 2019

CP Molecular Biology

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CRISPR/Cas9 technology enables the rapid generation of loss‐of‐function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR‐induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.

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“…The majority of Cas9 applications concern the cutting of one or both DNA strands within a gene body (Ran et al, 2013). Once the Cas9 protein can cause a nick or a double strand brake that is erroneously repaired by the DNA repair machinery, presence of Cas9 at the locus is neither necessary nor likely anymore (Brinkman et al, 2018). The story is however different for the activator VPR-dCas9.…”

Section: 5mentioning

confidence: 99%

Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

Schüller

Wolansky

Berger

et al. 2020

Preprint

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Current methods for forced expression of selected target genes are based on promoter exchange or on overexpressing native or hybrid transcriptional activators in which genespecific DNA binding domains are coupled to strong activation domains. While these approaches are very useful for promoters with known or synthetically introduced transcription factor binding sites, they are not suitable to turn on genes in biosynthetic gene clusters which often lack pathway-specific activators. To expand the discovery toolbox, we designed a Cas9based RNA guided synthetic transcription activation system for Aspergillus nidulans based on enzymatically disabled dCas9 fused to three consecutive activation domains (VPR-dCas9).Targeting two biosynthetic gene clusters involved in the production of secondary metabolites, we demonstrate the utility of the system. Especially in silent regions facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to our targeted promoters we have created a genome-wide nucleosome map to identify the cognate nucleosome-free-regions (NFRs). Based on these maps, different single-guide RNAs (sgRNA) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to activate silent BGCs in A. nidulans, partially to very high expression levels and also open the opportunity to stepwise turn on individual genes within a BGC that allows to decipher the correlated biosynthetic pathway.

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Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

Cited by 222 publications

References 59 publications

Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

Temporal analyses of CRISPR-directed gene editing on NRF2, a clinically relevant human gene involved in chemoresistance

Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

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