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Fig. 2 | Plant Methods

Fig. 2

From: Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Fig. 2

Screening by PCR and sequencing. Expected sgRNA cut indicated by ↓. Red text shows the sgRNA target sequence and bold text the PAM motif. Primary clones: Several primary clones contain sequences showing both CRISPR-induced mutations and the wildtype (WT) sequence, as seen by the presence of two bands following PCR, these are indicated by (+WT). Mutants M1–M4 were selected on plates and LM1 underwent selection in liquid media. M1 shows a 4 nt deletion from the second sgRNA whilst mutants M2–M4 show a 37 nt deletion between the two CRISPR-Cas cut sites. Sub-clones: The gel shows examples of a selection of sub-clones derived from the primary clones. Sub-clones are labelled according to the primary clone and sub-clone number. With the exception of M1_9, which gives a WT sequence and 4 nt deletion as seen in the primary clone, all sub-clones chosen for sequencing are bi-allelic. Two-thirds of sequenced bi-allelic sub-clones show a single sequence with a 37 nt deletion suggesting that both alleles carry the same mutation. In sub-clones where mutations differ between alleles both sequences are shown

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