"The entire human genome. Without a doubt, the most important and WONDROUS MAP ever produced..."

-Bill Clinton, June 2000


"Mapping the human genome has been compared with putting a man on the moon, but I believe it is more than that. This is the outstanding achievement not only of our lifetime, but in terms of human history... this code is the essence of mankind, and as long as humans exists, this code is going to be important and will be used." 


-Thomas Michael Dexter, June 2000


"Ever so often in the history of human endeavour, there comes a breakthrough that takes humankind across a frontier into a new era. ...  such a breakthrough, a breakthrough that opens the way for massive advancement in the treatment of cancer and hereditary diseases. And that is only the beginning."


-Tony Blair, June 2000



As envisioned by scientists and expressed by leaders, the mapping of the human GENOME has indeed opened up a universe of knowledge about our history, our relationship with this planet and its inhabitants, about life, and about disease. From the above quotes, it was clear that a "wondrous map" represented the beginning of an important revolution, and that this code would be used. 


Choosing coordinates for EPIGENETIC EDITING is only possible because GENOME maps now exist. For EPIGENOME understanding, however, thousands of genome maps, not only for humans, but for an ever-growing number of other species needed to be generated and compared. We now know which regulatory regions are conserved through evolution and by mapping EPIGENOME DATA to the GENOME sequence, genome-wide EPIGENETIC MAPS by cell or tissue type are being generated all the time and we really are beginning to understand how key developmental and diseases unfold on an EPIGENOMIC level. The use of these maps in choosing EPIGENETIC EDITING coordinates is outlined below. 

FINDING EPIGENOMIC COORDINATES TO ENGINEER

Where are you ENGINEERING? and What?

Unlike GENOME sequence, the EPIGENOME drastically changes over development. This enables highly choreographed gene expression control, which in turn facilitates differentiation into every cell type. Multi-institutional efforts such as ENCODE are providing "ROAD MAPS" for EPIGENETIC ENGINEERING. 

ENCODE and GENERATING ONES OWN EPIGENOMIC MAPS

Standing for the ENCYCLOPEDIA of DNA ELEMENTS, ENCODE seeks "to build a comprehensive parts list of functional elements in the human genome, including elements that act at the protein and RNA levels, and regulatory elements that control cells and circumstances in which a gene is active." In short, it represents an immense effort to collect key EPIGENOME DATA. This data is immensely useful in choosing candidate EPIGENETIC EDITING target sites, but for some targets new maps must be generated. For this, molecular biology tools and next generation sequencing technologies allow researchers to add new EPIGENOME MAPS, adding to our fundamental understanding of GENOME regulation and opening up new targets for EPIGENOME ENGINEERING.

crRNA design tools

Cas9 can be delivered to any coordinates by simple CRISPR or GUIDE RNA instructions. Based on a relatively simple set of rules, most genomic loci can be efficiently targeted. In short, virtually any 20 bp crRNA or gRNA sequence can be designed to complement any DNA sequence. Cas9 requires that the target sequence be followed by a special PROTOSPACER ADJACENT MOTIF (PAM) defined as any DNA base (N) followed by GG. As this PAM sequence is very common, virtually any gene or regulatory element in the genome can be targeted by designing a unique guide RNA. Many simple to use gRNA design tools are freely available now and many companies offer pre-designed and validated gRNA. 


Once selected, the gRNA and a universal tracrRNA are either cloned into a plasmid with a proper promoter for delivery to cells, or these can now be commercially produced and directly introduced to cell lines.


GRNA DESIGN TOOLS

Many gRNA DESIGN TOOLS now exist. In short, they offer a screening system of candidate gRNAs by organism GENOME. Positions on either strand are interrogated for PAM location and sequence uniqueness among the rest of the genome. Repetitive sequences are generally avoided.