To achieve high efficiency multiplexing with CRISPRm, we found it was necessary to modify the sgRNA design by including a 5′ HDV ribozyme, and to assay multiple RNA polymerase III promoters for a given strain background.
He was modifying the sample.
For this reason, we modified the junction sequence between U6 promoter and the DNA encoding sgRNA to see which variation could be acceptable.
To obtain a deletion with a precise fusion point, we modified the design of the ssODN targeting K08F4.2 to contain (1) 80- to 51-base homology arms that precisely flanked the sgRNA cut sites and (2) a restriction site inserted at the cut site (strategy 4 in Figure 1A and Figure S1D).
The epigenome modifies the genome.
oIK807 (5′-AGGAGCCACGGCATACATTC-3′, in the unc-22 locus) - oIK810 (5′-TGCCAATCTTTCTCGGACTTCTC-3′, in the unc-22 locus) PCR was performed to confirm that the unc-22 genomic locus was not modified through a sgRNA #1-mediated cut.
We also synthesized the chemically modified sgRNAs and CAS9 mRNA, which are more stable and shown to significantly improve genome-editing efficiency after directly transfecting into cells (Hendel et al., 2015).
I therefore tested whether this modified sgRNA(F+E) displayed increased activity in C. elegans, as a potential tool to improve editing efficiency.
Recent work in mammalian cells reported that a modified sgRNA(F+E), with an extended Cas9 binding structure and removal of a potential PolIII terminator by an A-U basepair flip, exhibits improved activity (Chen et al. 2013a).
The PU6:: unc-119 sgRNA plasmid (Friedland et al. 2013) was modified so that the NotI site was inserted between the K09B11.12 U6 promoter and the sgRNA backbone, deleting the unc-119 -specific sgRNA sequences.
