![]() Synchronized and released HCT 116–19 cells were electroporated with 24–120 pmol CRISPR/Cas9 RNP and 0.6–3.0 μM of 72mer. (A) Gene editing is dose dependent when directed by the RNP and the ssODN. A control digest was performed on the 345 base amplicon with the restriction enzyme DdeI. As a control, the RNP complex was incubated with an amplicon generated from the HBB gene 345 base pairs in length from cell line K562. In the complete reaction, two products were generated with sizes consistent with fragments predicted from the specific cut site designed for the RNP complex. The amplicon was combined with 25pmols and 50pmols of RNP complex respectively and incubated for 40 minutes at 37☌. Genomic DNA was isolated from untreated HCT 116–19 cells and PCR used to generate an amplicon of size 605bp, which surrounds the sequence of the integrated mutant eGFP gene. ![]() The lower section of the figure shows the 2C seed sequence and the tracrRNA sequence. Guide RNAs (gRNAs) direct and activate the Cas9 endonuclease which then cleaves the target DNA. Cas9 protein (gray) is added to complete RNP assembly. crRNA and tracrRNA are annealed in equimolar concentrations. crRNA provides target specificity (20 bases, red section) corresponding to the 2C protospacer sequence and an interaction domain (blue) with the tracrRNA (green). (A) CRISPR/Cas9 Ribonucleoprotein Assembly Reaction. The oligonucleotide used in these experiments is 72 bases in length bearing phosphorothioate modified linkages at the three terminal bases the 72-mer targets the non- transcribed (NT) strand (72NT). The highlighted bases in blue represent the 2C CRISPR protospacer sequence and the orange bases highlight the PAM site. The nucleotide targeted for exchange is bolded and underlined. The appropriate segments of the wild-type and mutated eGFP gene with the targeted codon, located in the center of the sequence, are displayed in green and red. Fixed a bug where SnapGene could hang after deleting an open file on OSX.Model system for gene editing of the mutant eGFP gene.Fixed a bug where SnapGene Viewer, if left running for an extended period of time, would not periodically check for updates.Fixed a bug with opening some GenBank files (Reported by Marius).Fixed a bug that could result in a crash when simulating PCR or any PCR based manipulation.Various color, text and alignment enhancements.Exported maps now use a white background.Improved circular plasmid identification. ![]()
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