A Survey of Validation Strategies for CRISPR-Cas9 Editing

Sentmanat, Monica F.; Peters, Samuel; Florian, Colin P.; Connelly, Jon P.; Pruett-Miller, Shondra M.

doi:10.1038/s41598-018-19441-8

Cited by 286 publications

(217 citation statements)

References 25 publications

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“…homozygous mutations). However, our analyses are unlikely to be affected by this T7E1 bias as they were performed on complex cell populations with maximum estimated efficiency rates of 30%, a range of activities in which the T7E1 assay can be considered a reliable estimation method (Sentmanat et al ., ). Our results, far from showing a clear front‐runner nuclease, indicated a strong target‐dependent efficiency.…”

Section: Discussionmentioning

confidence: 94%

Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

113

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Summary The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

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Section: Discussionmentioning

confidence: 94%

Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

113

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“…Several methods have been proposed to assess off‐targeting . T7E1 assay is generally used to validate gene cleavage using CRISPR/CAS9 system . In this study, results of T7E1 assay showed no cleaved products, indicating that there is a little off‐targeting effect.…”

Section: Discussionmentioning

confidence: 74%

SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells

et al. 2018

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There are no effective treatments for corneal endothelial diseases, except for corneal transplantation, as human corneal endothelial cells (hCECs) do not regenerate. The regeneration of hCECs could be induced through regulation of the expression of specific genes. In this study, we investigated whether the overexpression of sex‐determining region Y‐box 2 (SOX2) can regenerate hCECs in vivo and in vitro. SOX2 was activated using the clustered regularly interspaced short palindromic repeats (CRISPR)/deactivated CRISPR‐associated protein 9 (dCas9) activation system. Genes were transfected into the corneal endothelium of Sprague‐Dawley rats. Central corneal thickness and opacity were measured, and alizarin red S staining was performed. Corneal opacity and central corneal thickness were reduced in the SOX2 group compared with the control group. The density of CECs was higher in the SOX2 group compared with the control group. Additionally, hCECs were cultured and analyzed after overexpressing SOX2. Cell viability, proliferation rate, and the number of cells in S‐phase were increased after SOX2 overexpression (p < .05). Cyclin‐dependent kinase 1 and cyclin D1 were found to be overexpressed (p < .05). WNT signaling was repressed, and the AKT pathway was activated by SOX2 overexpression. Mitochondrial oxidative stress and energy production were increased by SOX2 overexpression (p < .05). In conclusion, SOX2 activation promotes wound healing and regeneration in CECs. SOX2 activation using the CRISPR/dCas9 system may thus be useful for the treatment of hCEC diseases. Stem Cells 2018;36:1851–12

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“…). Indels are typically small, no more than 1–15 nucleotides long . These apparently minor genetic changes are usually adequate to induce a crippling frameshift (indels of 1–2 nucleotides are most common) or premature stop codon, effectively knocking out gene function.…”

Section: Repurposing Crispr Proteins For Genome Editingmentioning

confidence: 99%

“…Indels are typically small, no more than 1-15 nucleotides long. 57,58 These apparently minor genetic changes are usually adequate to induce a crippling frameshift (indels of 1-2 nucleotides are most common) or premature stop codon, effectively knocking out gene function. Experimenters test the levels of editing at the target site by isolating the DNA from the cells or tissue and performing sequence analysis of amplicons through methods such as targeted next-generation sequencing, tracking of indels by decomposition, 59 or less reliable heteroduplex assays such as T7E1.…”

Section: Repurposing Crispr Proteins For Genome Editingmentioning

confidence: 99%

Clinical applications of CRISPR‐based genome editing and diagnostics

2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)‐driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR‐based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.

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A Survey of Validation Strategies for CRISPR-Cas9 Editing

Cited by 286 publications

References 25 publications

Assessment of Cas12a‐mediated gene editing efficiency in plants

Assessment of Cas12a‐mediated gene editing efficiency in plants

SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells

Clinical applications of CRISPR‐based genome editing and diagnostics

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