New high-throughput analysis reveals parameters affecting guideRNA efficiency

A recent report in Nature Methods by one of the leading geneticist, George Church, unveils factors that affect genome editing efficiency with CRISPR. The group from the Wyss Institute and Harvard Medical School employed a library-on-library approach to figure out why certain gRNAs are more effective than others. They carried out two separate experiments; first they transfected cells with a target lentiviral library as well as a gRNA plasmid library, which enabled the scientists to analyze how a certain sequence affect genome editing rates. Not quite surprising, there was a massive variation among different gRNAs at different targets, but the most important base was found to be the PAM adjacent base, with G being the most efficient and T least preferred. Next, they transfected the gRNA plasmid library into 293T cells and looked at endogenous genome editing. This experiment allowed to analyze the effect of epigenetic parameters on genome editing efficiency. Indeed, there was a robust correlation between DN-ase I hypersensitive sites (i.e. more open chromatin, transcriptionally active regions) and editing efficiency. Finally, it is interesting to note that there was no tangible relationship between gRNA effectiveness and off-target activity, moreover some of the most active gRNAs had the least off-target effects.

The group made a prediction algorithm available
online: https://crispr.med.harvard.edu/sgRNAScorer/

Protospacer Workbench - an offline software for for rapid sgRNA design and off-target prediction

An offline tool available for MacOS X system is now available for free at http://www.protospacer.com/. The software combines the advantages of various CRISPR design algorithms. It features single or paired target design of gRNAs for which cleavage efficiency is estimated based on the Doenech score and off-target sites are predicted and validated. The software can handle very large genomes, custom sequences or user defined genes. gRNA design is only available for S. pyogenes Cas9 for N(20)NGG sites. It has a graphical user interface and does not require programming skills.  The workbench also connects to the IGV viewer.
Source: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3291.html

Chemically modified guide RNAs facilitate genome editing in primary human cells 

A recent work from the Porteus lab (Stanford, California) presents the use of chemically altered gRNAs to improve low efficiency genome editing rates in primary human T cells and CD34+ hemopoietic stem cells. Modified sugar-phosphate backbone renders the gRNA more stable in cells leading to enhanced gene engineering, including more efficient non-homologous end joining and homology directed repair rates. These gRNAs have a particular advantage over unmodified gRNAs when the system is delivered as a ribonucleoprotein particle (RNP, i.e. Cas9 protein and gRNA) or as pure RNAs (Cas9 mRNA and gRNA), advantages over plasmid DNA expressed Cas9 are less clear. The authors claim there is somewhat better on/off target ratio with modified gRNAs, but we have to be cautious with this: the authors modified the terminal bases on the gRNA, perhaps functionally truncating them. Truncated gRNAs have significantly higher specificity according to the paper of from the K. Joung lab. Nevertheless, this system could be highly advantageous when working with primary cells, where genome editing rates are relatively low.

If you are interested in the paper, you can find it here.