Precision Medicine, CRISPR, and Genome Engineering Moving from Association to Biology and Therapeutics

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getting a glimpse of the whole genome, the whole epigenome, the whole transcrip-

tome, and the whole proteome. This technological development—in many cases

driven by next generation sequencing (NGS)—has helped create a comprehensive

parts-list. In most cases, though, we still lack an understanding of the connections

between each of the parts.

The CRISPR/Cas9 (Clustered Regular Interspaced Palindromic Repeats/CRISPR-

Associated System 9) adaptive immune system in bacteria and archaea has provided

a simple and efficient means of site-specifically modifying genomes of interest.

Applications of the technology, discussed herein, hold the potential to push our

understanding of development beyond the parts (reductionism) toward an

understanding of how complex phenotypes emerge from the hierarchical and inter-

dependent connections between these parts (holism). Studies highlighted illustrate

Cas9

**NGG

HNH

RuvC

dCas9

**

NHEJ

**

Donor Template

Imprecise

Precise

HDR

INDEL

A

C

D

HNH

RuvC

NGG

Fig. 3.1 An old idea meets a new technology. (a) Waddington’s Epigenetic Landscape. A ball,
representative of a developing cell, is pulled through one of many developmental pathways to
reach the bottom of the hill as a mature, differentiated cell. (b) Waddington envisioned that net-
works of genes and their products shaped the landscape. The black boxes represent genes and the
lines, the gene products. (c) A schematic of the CRISPR Cas9/guide RNA complex. Cas9 contains
two endonuclease domains (HNH and RuvC) that generate a double-strand break positioned three
nucleotides upstream of the Cas9-specific PAM, NGG (Left). When these nuclease domains are
mutated, dead Cas9 (dCas9) no longer generates DNA breaks, but rather serves as a scaffold to
recruit additional protein domains (depicted in green) that can modify the epigenome. (d) Two
types of repair can follow Cas9-induced breaks. Repair by non-homologous end joining (NHEJ)
results in imprecise repair and the inclusion of insertion and/or deletions (Indels). Repair by
homology-directed repair (HDR) using a co-delivered donor template results in precise genomic
modifications (in green). Figure 3.1a, b is reprinted from [ 2 ] with permission from The Taylor and
Francis Group

3 From Reductionism to Holism: Toward a More Complete View of Development...