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Featured CRISPR Based Base Changes

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  1. Nov 6, 2017 #1

    BillTre

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    CRISPR is often used to switch out a length of DNA for a different piece of sequence, which can change several base pairs at once.
    Here is a Science news report on how researchers have now developed methods to efficiently change single base pairs in a sequence (or single bases in single stranded RNAs).
    It can also work well in non-dividing cells. Normally CRISPR does not efficiently switch lengths of sequence in non-dividing cells because the switching mechanism depends on the cell's homology directed repair mechanism, which is only active in dividing cells.
     
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  3. Nov 7, 2017 #2

    Ygggdrasil

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    Here are citations for the papers discussed in the Science news piece:
    Gaudelli et al. 2017 Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature Published online 25 Oct 2017. doi:10.1038/nature24644

    Cox et al. 2017 RNA editing with CRISPR-Cas13. Science Published online 25 Oct 2017 doi:10.1126/science.aaq0180

    Main drawback of the two techniques is the limited sequence specificity of the enzymes used to edit DNA/RNA. The ADAR enzyme used in the Science study can only be used to cause the equivalent of A-->G mutations in RNA and the enzyme works best on A bases only in certain sequence contexts. The editing efficiency was as high as ~ 40% on optimal substrates for the ADAR enzyme, but as low as 10% on non-optimal substrates. Substantial work will be needed improve these editing efficiency numbers (though this may not be necessary for all applications) and expand the types of base changes the system can perform.

    The DNA editing system described in the Nature paper catalyzes only the conversion of an A-T pair to a G-C pair, which complements previous work by the same group and others allowing the opposite conversion. This is quite impressive because no natural enzymes catalyze this type of base conversion, so the researchers had to engineer a new enzyme to perform that particular change. Other types of mutations are not yet possible, though it may be possible to engineer new enzymes for other types of conversions (some of the directed evolution methods from the Nature paper could help engineer new RNA editing enzymes as well). The editing efficiency is only ~ 50%, but this is comparable to the efficiency with which CRISPR can create site-specific mutations. CRISPR is still more flexible in terms of the types of mutations it can introduce and more work needs to be done to see which system introduces fewer unwanted off-target mutations.
     
    Last edited: Nov 7, 2017
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