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Fig. 2 | Plant Methods

Fig. 2

From: Meeting report: GARNet/OpenPlant CRISPR-Cas workshop

Fig. 2

Detection of induced mutations. a If two single guide RNAs were delivered with the aim of deleting a fragment of DNA, oligonucleoitide primers flanking the targets can be used to PCR amplify the locus. Evidence of an amplicon, smaller that that obtained in a wildtype (WT) control is indicative of a deletion. The absence of an amplicon of equivalent size to the WT may indicate a homozygous deletion. b If the quantity of the deletion amplicon is low or absent, the genomic DNA can be digested with any restriction endonuclease (REN) with one or more recognition sites in the deletion region prior to PCR amplification. This will remove any wild-type sequence enabling the detection of deletions even if at low quantity in the sample. c Double strand breaks (DSBs) are most likely to occur three base pairs before the PAM in the seed-region of the target. Small insertion-deletion events at the target can be detected by digesting a PCR amplicon of the target locus with a REN for which the cognate sequence would be disrupted by imperfect repair of the DSB

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