Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing

Lomov, Nikolai; Viushkov, Vladimir S.; Petrenko, A P; Syrkina, M. S.; Rubtsov, Mikhail A.

doi:10.1134/s0026893319060116

Cited by 12 publications

(11 citation statements)

References 40 publications

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“…All 20 mL of digested heteroduplexes was run on a 2% agarose gel stained with ethidium bromide, and the 100-bp DNA ladder was run with the sample for reference. 26 RNA extraction, reverse transcription, and quantitative PCR Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following standard RNA extraction protocols. First-strand cDNA was synthesized from the isolated RNA with SuperScript IV Reverse Transcriptase (Invitrogen).…”

Section: Knock-out Of the D2hgdh Gene In Car-t Cells Via Crispr/ Cas9...mentioning

confidence: 99%

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment

et al. 2022

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Section: Knock-out Of the D2hgdh Gene In Car-t Cells Via Crispr/ Cas9...mentioning

confidence: 99%

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment

et al. 2022

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“…It can be applied either as an end-point fluorescence reaction (e.g., T7E1, Surveyor, RFLP, CAPS) or as a quantitative approach, where the amplification curve is monitored. These offer the ability to illuminate guide RNA efficacy by distinguishing mutant from wild-type cells (Zischewski et al 2017;Lomov et al 2019). Capillary PCR fragment size analysis can also be used to detect differences as small as a few base pairs.…”

Section: Pcrmentioning

confidence: 99%

“…Depending upon the size of the edit, PCR may be followed by polyacrylamide or agarose gel-based visualization (a few base pairs up to kilobases). The band intensities can provide information regarding the ratio of mutated to unmutated cells (Lomov et al 2019).…”

Section: Pcrmentioning

confidence: 99%

Detection of genome edits in plants—from editing to seed

Shillito

Whitt

Ross

et al. 2021

In Vitro Cell.Dev.Biol.-Plant

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Genome editing (also known as gene editing) employs a range of tools such as Meganucleases, Zinc Finger Nucleases, TALENs, and more recently CRISPR to make defined changes in genes, regulatory sequences, untranslated regions, or intergenic regions. It is increasingly being applied in plant science research and to improve plant varieties. The benefits of having effective detection tools begin with optimization of the genome editing process itself and continue with selection and characterization of tissue cultures and/or regenerated plants. Detection tools are also used throughout the breeding process, and for preparation of regulatory dossiers when required, as well as for seed production, and may be necessary for monitoring products in the marketplace. Detection and identification of genome edits employs a wide range of analytical approaches including PCR, digital PCR, and sequencing methods. This article examines the applicability of each category of detection or identification approach, from the optimization of genome editing processes, through creation of edits, selection and characterization, and breeding. The challenges surrounding the detection of genome edits present at low levels in large seed, plant, or grain populations and of differentiating directed genome edits from conventional mutations are also explained.

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“…As in any mutagenesis technique, false positives of NBT are numerous, while reproducibility is not the topical aim of biotechnologists (Bortesi and Fischer, 2015;Lomov et al, 2019). The reality of the transformation must be verified because somaclonal variation can, in some cases, account for the observed phenotype (e.g., tolerance to an herbicide) (Anderson et al, 2008;Birch, 1997;Liu et al, 2012;Potrykus, 1991).…”

Section: The Issue Of False Positivementioning

confidence: 99%

Advances in identifying GM plants: toward the routine detection of ‘hidden’ and ‘new’ GMOs

Bertheau¹

2021

Burleigh Dodds Series in Agricultural Science

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In 2018 the Court of Justice of the European Union recalled that organisms with genomes modified by artifactual techniques should be considered GMOs under European regulations. GMOs derived from cultures of cells isolated in vitro or from new genomic techniques must therefore be traceable. This chapter reviews the various technical steps and characteristics of those techniques causing genomic and epigenomic scars and signatures. These intentional and unintentional traces, some of which are already used for varietal identification, and are being standardized, can be used to identify these GMOs and differentiate them from natural mutants. The chapter suggests a routine procedure for operators and control laboratories to achieve this without additional costs.

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Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing

Cited by 12 publications

References 40 publications

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment

Detection of genome edits in plants—from editing to seed

Advances in identifying GM plants: toward the routine detection of ‘hidden’ and ‘new’ GMOs

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