Modified Guide-RNAs Enhance CRISPR Editing

Hendel et al. (2015) have shown in Nature Biotechnology that certain chemical modifications to guide RNAs can enhance mutagenesis frequencies of the CRISPR/Cas9 system.  Clearly, these types of enhancements, should they pan out to be effective beyond human cells, could be of great interest to insect biologists.

guide RNA relative to target sequence

guide RNA relative to target sequence

Guide RNAs in the CRISPR/Cas9 system for genome editing are 100 nt long.  Twenty nucleotides at the 5′ end are complementary to the target sequence.  The sequences 3′ of the target-hybridizing region forms a double-stranded structure that is important for interacting with the Cas9 protein.

There is variability in the effectiveness of the system based on what cells are being mutagenized.  For example, the system works very well human tissue culture cells and less well in primary human cells.  Consequently, finding performance enhancements could be important.

Hendel et al. chemically synthesized guide RNAs and incorporated modified nucleotides at the 5′ and 3′ terminal positions of the guide.  Three chemical modifications were tested, 1) 2′-O-methyl (= M), 2) 2′-O-methyl 3′ phosphorothioate (=MS) and 3) 2′-O-methy 3’thioPACE (phosphonoacetate)(=MSP).

RNAThey proceeded to test 3 different guide RNAs, each with each of the 3 modifications in human cell lines (K526 cells) and in primary cells (T cells and hematopoietic stem progenitor cells, HSPC).

They made a number of interesting observations.  Their basic finding was that all modified forms of the guide RNA were better than unmodified guide RNAs in terms of the frequency of indel formation.  Generally speaking it seemed that the MSP modified guide RNA had an overall superior performance compared to the other modified forms.

The authors also report on using different modes for delivering Cas9 protein – plasmid DNA, mRNA, protein.  As others have also shown in a number of systems, transient expression of Cas9 mRNA from a plasmid was the least effective means by which to generate Cas9 protein in cells.  Transfecting mRNA or ,better yet, preassembled Cas9 protein and guide RNAs was much more effective.  Working with the Cas9 protein seems to be the approach that is yielding the highest levels of mutagenesis under a variety of conditions and in a number of systems.

What makes these modified guides more effective?  This is not addressed directly in the manuscript but the authors did measure the effects of modifications on the half-life of the guide RNAs and found the the MS and MSP forms showed significant enhancements in their stability.  They saw essentially no change in the activity of MSP modified guide RNAs 24 hrs after transfections whereas unmodified guides appear to be gone within a few hours.

So overall there were a number of benefits to using chemically synthesized and modified guide RNAs – increased efficacy, scalable production (which will be of interest to those thinking about therapeutics), more design options compared to transcribed guides.  Of course, a down-side might be cost.

This is an interesting paper that is worth reading if you are working with or thinking about CRISPR-based gene editing.

Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E. et al., 2015 Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotech advance online publication: doi: 10.1038/nbt.3290

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