Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

Haapaniemi, Emma; Botla, Sandeep K.; Persson, Jenna; Schmierer, Bernhard; Taipale, Jussi

doi:10.1101/180943

Cited by 17 publications

(23 citation statements)

References 18 publications

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“…We also demonstrated that wild-type p53 does not appreciably restrict targeted knockin by tandem paired nicking. In contrast, when a Cas9-nuclease-based method was applied, targeted knockin preferentially occurred in p53-suppressed cells as described previously (Haapaniemi et al, 2018;Ihry et al, 2018). A concern raised here is that when using Cas9 nuclease, designed genetic modification might be preferentially introduced into cells with compromised p53 function, which can spontaneously emerge within cultured cells.…”

Section: Discussionmentioning

confidence: 87%

“…p53 Does Not Significantly Affect Targeted Knockin by Tandem Paired Nicking It was recently shown that Cas9-nuclease-based genome editing triggers the activation of the p53 signaling pathway and G1 cell cycle arrest. As targeted knockin is mediated by HDR, which predominantly occurs in the S through G2 phases, Cas9 nuclease inherently obstructs targeted knockin in a p53-dependent manner (Haapaniemi et al, 2018;Ihry et al, 2018).…”

Section: Tandem Paired Nicking Does Not Trigger a Significant Level Omentioning

confidence: 99%

“…In addition, CRISPR/Cas9 often triggers unexpected cleavage followed by indel formation at off-target genomic sites (Fu et al, 2013;Hsu et al, 2013;Pattanayak et al, 2013;Ran et al, 2013a). Furthermore, it was recently shown that DSBs induced by CRISPR/ Cas9 activate the p53 signaling pathway and that wild-type p53 inhibits homology-directed genome engineering by CRISPR/Cas9 (Haapaniemi et al, 2018;Ihry et al, 2018). Technical improvements to overcome these issues, in addition to the maintenance of substantial efficiency of targeted knockin, are requisites to develop a method for precise and efficient genome engineering.…”

Section: Introductionmentioning

confidence: 99%

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Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

et al. 2020

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Graphical Abstract Highlights d Tandem paired nicking promotes targeted knockin with substantial efficiency d Tandem paired nicking scarcely creates indels at the edited genomic loci d DNA recombination elicited by tandem paired nicking is analogous to Holliday's model d Targeted knockin by tandem paired nicking shows no reciprocal interference with p53 SUMMARYTargeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday's model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

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Section: Discussionmentioning

confidence: 87%

Section: Tandem Paired Nicking Does Not Trigger a Significant Level Omentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

et al. 2020

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“…S4E), and P21 sgRNAs did not enrich in H1-hESCs. In contrast, in a genome-scale screen in retinal pigment epithelial cells, where Cas9 activity causes TP53-dependent cell cycle arrest rather than apoptosis, sgRNAs targeting TP53 and P21/CDKN1A were enriched while PMAIP1 sgRNAs were not (File S5) (Haapaniemi et al, 2017). Overall, these results indicate that PMAIP1 plays a role in the sensitivity of hPSCs to DNA damage and highlights the ability of genome-scale CRISPR screens to identify cell type-specific genes important for the fitness of pluripotent stem cells.…”

Section: Resultsmentioning

confidence: 99%

Genome-Scale CRISPR Screening Identifies Novel Human Pluripotent Gene Networks

Ihry¹,

Salick²,

Ho³

et al. 2018

Preprint

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15Human pluripotent stem cells (hPSCs) generate a wide variety of disease-relevant cells that can 16 be used to improve the translation of preclinical research. Despite the potential of hPSCs, their 17 use for genetic screening has been limited because of technical challenges. We developed a 18 renewable Cas9/sgRNA-hPSC library where loss-of-function mutations can be induced at will. 19Our inducible-mutant hPSC library can be used for an unlimited number of genome-wide 20 screens. We screened for novel genes involved in 3 of the fundamental properties of hPSCs: 21Their ability to self-renew/survive, their capacity to differentiate into somatic cells, and their 22 inability to survive as single-cell clones. We identified a plethora of novel genes with unidentified 23 roles in hPSCs. These results are available as a resource for the community to increase the 24 understanding of both human development and genetics. In the future, our stem cell library 25 approach will be a powerful tool to identify disease-modifying genes. 27 VISUAL ABSTRACT 28All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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“…programmable endonucleases that drive random or site-specific integration, respectively (Ivics et al, 2009;Mikuni et al, 2016;Suzuki et al, 2016). Transposases provide the highest efficiency of integration, a process that they directly mediate, whereas endonucleases only indirectly stimulate transgene insertion through the host repair machinery upon target locus cleavage, with potential detrimental side effects (Haapaniemi et al, 2018). Transposon-based systems achieve high rates of integration with >1-kb-long sequences, can handle cargos up to 100 kb (Jung et al, 2016;Li et al, 2011), and are thus ideal for demanding applications requiring integration of one or multiple large transgenes comprised of a promoter and gene of interest (GOI).…”

Section: Accessmentioning

confidence: 99%

Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch

Kumamoto

Maurinot

Barry‐Martinet

et al. 2020

Neuron

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Summary Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro , iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo , we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.

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Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

Cited by 17 publications

References 18 publications

Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53

Genome-Scale CRISPR Screening Identifies Novel Human Pluripotent Gene Networks

Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch

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