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Nuclease resistance improves performance of synthetic CRISPR RNA with Cas9 mRNA
When development of the Edit-R predesigned crRNA product line began back in 2013, our goal was to design and manufacture an optimal, predesigned, genome-wide product line for CRISPR-mediated knockout. One major accomplishment towards this goal was our development of a data-driven algorithm for selecting guide RNAs more likely to cause functional protein knockout, not just create a double-strand break. For 2017, we are drawing on our RNA synthesis expertise to further improve experimental outcomes for our customers.
Figure 1 - A. Position of modifications for nuclease resistance on Edit-R synthetic crRNA and tracrRNA. Edit-R synthetic guide RNAs contain 2ʹ-O-methyl (2ʹ-OMe) and backbone phosphorothioate linkages (PS) on the two nucleotides at the 5ʹ end of the crRNA (green strand) and on the two nucleotides at the 3’ end of the tracrRNA (blue strand). B. Structure of the 2ʹ-OMe and PS backbone phosphorothioate linkages.
With the advent of Cas9 mRNA and protein reagents, we found that CRISPR efficiency in some applications, especially co-electroporation with Cas9 mRNA, could be improved when the crRNA and tracrRNA were modified for improved stability (nuclease-resistance). RNA modifications to resist degradation are not new to the field, nor to Dharmacon products. Our siSTABLE™ siRNA modification pattern and Accell™ siRNA reagents both include modified bases and backbone linkages for improved nuclease resistance.
We systematically tested modifications on the 2ʹ position of the sugar and phosphate backbone of both crRNA and tracrRNA. We found several positions on both the crRNA and tracrRNA where modifications were not well-tolerated and led to a decrease in editing efficiency. In addition, we found that having more than two 2’-O-methyl bases and phosphorothioate linkages in specific patterns on the crRNA and tracrRNA were very toxic in some cell types. When our selected pattern of stabilizing modifications is applied to the single-strand region of the crRNA:tracrRNA complex (Figure 1) co-electroporation results with Cas9 mRNA (Figure 2A) are greatly improved, and to some degree, Cas9 protein co-electroporation results are also improved (Figure 2B).
Table 1 illustrates the overall benefits of stabilized Edit-R synthetic crRNA and tracrRNA across different CRISPR-Cas9 gene editing applications. Virtually no difference in editing efficiency is detected when modified or unmodified crRNA:tracrRNA is transfected into a Cas9-expressing cell line or co-transfected with Cas9 protein as an RNP complex. However, co-transfection and co-electroporation with Cas9 mRNA are improved by the nuclease resistant properties of these modifications.
Figure 2 - A. Modifications to block degradation by nucleases are required for successful co-electroporation of Cas9 mRNA and crRNA:tracrRNA. B. Co-electroporation of Cas9 protein and crRNA:tracrRNA does not require stabilizing modifications, but results may be improved with their addition.
As of February 2017, all Edit-R predesigned crRNA and tracrRNA will carry these modifications for nuclease resistance with no change in price! Custom crRNA designed and ordered with the CRISPR RNA Configurator will also be modified by default, but unmodified is still an option. Edit-R tracrRNA (Cat # U-002005-xx) will carry the 3ʹ modification for nuclease resistance with no change in price. If you currently have some unmodified crRNA or tracrRNA (Cat# U-002000-xx) in your freezer, not to worry! Unless you are working with an application that requires nuclease stability (see Table 1), you can use any combination of modified or unmodified Edit-R crRNA and tracrRNA.
If you have any questions about using Edit-R CRISPR RNA in your lab, please contact our Technical Support team.
These publications demonstrate Edit-R CRISPR-Cas9 genome editing reagents for target gene knockout or precise knock-in in a variety of applications.
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