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For CRISPRa experiments, what if a gene has more than one transcriptional start site?
The published CRISPRa v2 algorithm (Horlbeck et al., 2016) used FANTOM and Ensembl databases to predict more accurately the transcriptional start site (TSS). Some genes (6.8%) were identified as having alternative transcriptional start sites. The publication lists 10 gRNA designs per TSS. For the Edit-R predesigned guide RNAs, we offer the top four guide RNAs for the primary TSS, and, when applicable, four guide RNAs for the secondary TSS. These are labeled as P1 and P2, respectively. If the CRISPRa guide RNAs for your gene do not have a P2 designation, then only a single start site is designated for that gene. If your gene has both P1 and P2 guide RNAs, it might be beneficial to test both for your experiment. Which TSS is active and to what level depends on your cell line. For a small number of genes (0.1%) Horlbeck et al., 2016 identified more than two TSS. We only offer P1 and P2 designs as catalog items, but we can generate the additional guide RNAs as a custom request based on designs from the published algorithm.