Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering

Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5’ fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering. DOI: http://dx.doi.org/10.7554/eLife.25312.001
eLife digest There are several different technologies that can be used to make specific changes to particular genes in cells. These “gene editing” approaches have the potential to help humans in a variety of different ways, for example, to treat diseases that presently have no cure, or to improve the nutritional quality of crop plants. One such gene editing approach is known as CRISPR. To edit a specific gene, a molecule called a guide ribonucleic acid (or guide RNA for short) binds to a section of the gene and recruits an enzyme to cut the DNA encoding the gene in a particular location. Adding a “donor” DNA molecule that contains the desired “edit” can lead to the cell repairing the broken gene in a way that incorporates the desired change. Modifying the guide RNA or the donor DNA can enhance CRISPR editing. For example, extending the guide RNA molecules by adding “aptamer” sequences can enable researchers to specifically activate the genes that have been edited. It is also possible to add chemical tags to RNA and DNA, but it is not clear how chemical modifications to the guide RNA and donor DNA could affect CRISPR. Here Lee et al. investigated whether adding chemical tags to the guide RNA and/or donor DNA could enhance gene editing. The experiments show that the modified guide RNAs and donor DNAs were still active and could edit DNA in mouse and human cells. Adding a fluorescent molecule to the donor DNA allowed Lee et al. to track which cells contained donor DNA and separate them from other cells. The fluorescent cells had twice as much editing compared to groups of unsorted cells. In further experiments, the guide RNA and donor DNA were fused together and supplied to cells together with a DNA cutting enzyme. Cells containing this combined molecule had three times more editing than cells exposed to the original CRISPR system. This change may aid the development of new uses for CRISPR because it simplifies the system from three components (an enzyme, guide RNA and donor DNA) to just a cutting enzyme and the combined molecule. Overall, the findings of Lee et al. show that chemical modifications to guide RNA and donor DNA can make the CRISPR system more versatile. It opens up the possibility of new applications such as adding a targeting group that would direct the CRISPR Cas9 system to a specific cell type or tissue. DOI: http://dx.doi.org/10.7554/eLife.25312.002