CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

Chylinski, Krzysztof; Hubmann, Maria; Hanna, Ruth E.; Yanchus, Connor; Michlits, Georg; Uijttewaal, Esther C. H.; Doench, John G.; Schramek, Daniel; Elling, Ulrich

doi:10.1038/s41467-019-13403-y

Cited by 33 publications

(47 citation statements)

References 43 publications

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“…These numbers are in accordance with previous mathematical This strategy resulted in improved editing efficiencies. This strategy could be further optimized and de-risked for clinical application by developing inducible Pol III promoters which restrict HIV-1 gRNAs expression to HIV-1-infected cells [44][45][46][47] .…”

Section: Discussionmentioning

confidence: 99%

Durable Control of HIV-1 Using aStaphylococcus aureusCas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

Chávez¹,

Reddy²,

Raymond³

et al. 2020

Preprint

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CRISPR/Cas9 gene editing has the potential to revolutionize the clinical management of HIV-1 infection, and may eliminate the need for antiretroviral therapy (ART). Current gene therapies attempt to either excise HIV-1 provirus or target HIV-1 entry receptors to prevent infection of new cells. Using a viral dynamic model, we determined that combining these two interventions, in the presence or absence of ART, significantly lowers the gene editing efficacy thresholds required to achieve an HIV-1 cure. To implement this dual-targeting approach, we engineered a single lentiviral vector that simultaneously targets multiple highly-conserved regions of the provirus and the host CXCR4 coreceptor, and developed a novel coculture system enabling real-time monitoring of latent infection, viral reactivation, and infection of new target cells. Simultaneous dual-targeting depleted HIV-1-infected cells with significantly greater potency than vectors targeting either virus or host independently, highlighting its potential as an HIV-1 cure strategy.

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Section: Discussionmentioning

confidence: 99%

Durable Control of HIV-1 Using aStaphylococcus aureusCas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

Chávez¹,

Reddy²,

Raymond³

et al. 2020

Preprint

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“…Finally, the 3C landing site line would serve as the best possible control to a 3C landing site gene inactivation line because both lines utilize the same transgene integration site. Another highly efficient system, termed CRISPR-Switch, also allows Cre-controlled CRISPR/Cas9 mutagenesis (Chylinski et al, 2019). However, in contrast to 3C mutagenesis, which enables the expression of Cas9 following a Cre recombination event, CRISPR-Switch controls the production of the gRNA in a Cre-dependent manner.…”

Section: Discussionmentioning

confidence: 99%

Cre-Controlled CRISPR (3C) Mutagenesis: Fast and Easy Conditional Gene Inactivation in Zebrafish

Hans¹,

Zöller²,

Hammer³

et al. 2020

Preprint

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Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects. The most commonly used technique to achieve conditional gene inactivation employs the Cre/loxP system and its ability to delete DNA sequences flanked by two loxP sites. However, targeting critical exons or an entire gene with two loxP sites is time and labor consuming. To circumvent these issues, we developed Cre-Controlled CRISPR (3C) mutagenesis. 3C mutagenesis is simple, fast and allows gene inactivation in a Cre-dependent manner. In contrast to loxP-flanked alleles, the recombined cells become fluorescently visible enabling the isolation of these cells and their subjection to various omics techniques. Moreover, 3C will be scalable and will enable the conditional inactivation of multiple genes simultaneously. Hence, 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles and should be applicable to all model organisms amenable to single integration transgenesis.

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“…[ 101,102 ] Furthermore, Cas9/gRNA modifications allow for chemical‐induced entry to the nucleus, light‐switchable enzyme activation, and Cre‐switchable gRNAs, for strict temporal control of editing. [ 103–106 ]…”

Section: Techniques To Mitigate Off‐target Cleavagementioning

confidence: 99%

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On‐ and Off‐Target Damage

2020

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Large deletions and genomic re‐arrangements are increasingly recognized as common products of double‐strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats ‐ CRISPR associated protein 9 (CRISPR/Cas9) on‐target sites. Together with well‐known off‐target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9‐editing is necessary to ensure validity of observed phenotypes in Cas9‐edited cell‐lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide‐RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off‐targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on‐ and off‐target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.

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CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

Cited by 33 publications

References 43 publications

Durable Control of HIV-1 Using aStaphylococcus aureusCas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

Durable Control of HIV-1 Using aStaphylococcus aureusCas9-Expressing Lentivirus Co-Targeting Viral Latency and Host Susceptibility

Cre-Controlled CRISPR (3C) Mutagenesis: Fast and Easy Conditional Gene Inactivation in Zebrafish

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On‐ and Off‐Target Damage

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