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Fig. 4 | Mobile DNA

Fig. 4

From: Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9

Fig. 4

Screening effective sgRNAs for CRISPR-Cas9 mediated knockout of the KRAB-Zfp gene cluster(s). A A diagram illustrates the methodology of a CRISPR-Cas9 based reporter assay for screening the effective sgRNAs with multi-targets. SgRNAs that target to the cluster-specific linker sequences of KRAB-Zfps were designed and cloned into pX459, respectively. Their targeting sequences (including the corresponding PAM sites) were arranged in an array and fused together with the mCherry encoding sequences (PEST was added to accelerate the turn-over rate of the mCherry protein) and the entire open reading frame was ligated into the mammalian expression vector, pcDNA3.1. B Testing the screening assays in (A) with an empty pX459 vector (a negative control) and two positive sgRNA controls against Zfp809. The representative fluorescence image for each transfection was shown on top. The images were pseudo-colored to facilitate the recognition of some special readers. The bar graph at the bottom demonstrates the measured fluorescence changes of mCherry upon the expression of Cas9 and the indicated positive control sgRNAs. C A diagram demonstrates the target specificity and the numbers of the predicted cleavage sites of the sgRNAs specifically designed for the indicated KRAB-Zfp cluster(s). The color intensity is proportional to the number of the predicted cleavage sites. D The fluorescence changes of the mCherry reporter upon the expression of Cas9 together with the indicated sgRNAs listed in (C). The statistical analyses were carried out to compare each sgRNA’s effect to a negative control with an empty pX459 plasmid being transfected. “**” stands for an extremely significant change (p-value is smaller than 0.01); “*” stands for a significant change (p-value is greater than 0.01 but smaller than 0.05); n.s. stands for no significant change (p-value is greater than 0.05)

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