From Reductionism to Holism: Toward a More Complete View of Development Through Genome Engineering

Abstract

Paradigm shifts in science are often coupled to technological advances. New techniques offer new roads of discovery; but, more than this, they shape the way scientists approach questions. Developmental biology exemplifies this idea both in its past and present. The rise of molecular biology and genetics in the late twentieth century shifted the focus from the anatomical to the molecular, nudging the underlying philosophy from holism to reductionism. Developmental biology is currently experiencing yet another transformation triggered by '-omics' technology and propelled forward by CRISPR genome engineering (GE). Together, these technologies are helping to reawaken a holistic approach to development. Herein, we focus on CRISPR GE and its potential to reveal principles of development at the level of the genome, the epigenome, and the cell. Within each stage we illustrate how GE can move past pure reductionism and embrace holism, ultimately delivering a more complete view of development.

Keywords: CRISPR; Conrad H. Waddington; Development; Epigenome; Genome; Genome engineering; Holism; Reductionism.

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 networks 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

Fig. 3.2

Pooled high-throughput CRISPR GE screens. A schematic details the steps involved in pooled, high-throughput CRISPR GE screens. (1) Large-scale production of guide RNAs in situ is followed by bulk cloning into a desired vector to generate a gRNA library. (2) The library is packaged in virus and used to infect a population of cells at a low multiplicity of infection (MOI) to avoid infection of a single cell with multiple gRNA plasmids. (3) Treatment of cells to induce a phenotype of interest, (4) followed by selection for the phenotype results in a population of cells enriched for gRNAs that contribute to the phenotype and depleted of those that do not. (5) Deep-sequencing of the selected population in comparison to the initial population reveals changes in the relative enrichment and/or depletion of gRNAs, suggesting genes involved in the phenotypic network. Figure adapted from relevant publications

Fig. 3.3

Epigenome modifications with CRISPR GE. (a) A schematic depicts the recruitment of dCas9 fused to activation or repression domains to effect changes in gene expression. The activating and repressing modules that have been used are displayed. (b) A schematic depicts the recruitment of dCas9 fused to catalytic domains that incorporate (right) or remove (left) epigenetic modifications. The enzymes (or catalytic domains) that have been used alongside dCas9 are listed, including their targeted modification and whether they work to add (+) or remove (−) the mark. (c) Several alternative scaffolds beyond direct protein fusion to Cas9 have been employed. The SunTag makes use of single-chain variable antibody-epitope interactions to recruit several functional moieties to a single molecule of dCas9. Modifications of the gRNA to include aptamers, such as the MS2 and PP7 hairpins, can be used to recruit functional domains to the gRNA, itself, preserving dCas9 as a neutral partner. This allows the targeting of distinct functionalities to different genomic loci simultaneously. Finally, dual recruitment through both dCas9-fusions and gRNA-aptamer scaffolds has been used to enhance the effects of the recruited functionality and recruit distinct moieties to a single genomic locus. (d) Dead Cas9-fusions with fluorescent molecules have been used to visualize genomic loci in fixed and live cells. Tethering using dCas9 has not yet been demonstrated, but could conceivably be used to site-specifically recruit transcription factors (TF) of interest and/or force interactions between distal genomic loci with dCas9 molecules harboring hetero-dimerization domains

Fig. 3.4

Lineage tracing with CRISPR GE. (a) A schematic depicts an idealized example of lineage tracing with Cas9. An array of CRISPR targets is inserted into the genome and subject to the activity of the introduced Cas9/gRNA complex. Mutations induced by Cas9 within the array are replicated and maintained throughout cell division. Thus, the CRISPR array of a mature cell serves as a memory of all Cas9 events that occurred throughout development and acts as a unique barcode signifying its developmental history, or lineage. The relationships between these barcodes (determined by NGS) can then be used to reconstruct a lineage map. (b) An example of an inter-target deletion, or ‘dropout.’ In the first round of CRISPR-mediated DSB and repair, only the fourth target is modified (change in color to green). However, during the second round, Cas9 induces DSBs in both the third and fifth target, leading to a deletion of the previously modified fourth target. This dropout event results in a loss of information. Red arrowheads depict DSB induction. (c) An example of a homing or self-targeting gRNA. The sequence of the gRNA is engineered to contain a PAM site between the spacer and scaffold portions of the gRNA, thus allowing the gRNA to target the locus from which it was derived. Multiple rounds of self-targeting result in the accumulation of mutations within the spacer sequence. A single round is shown with the induced mutation depicted as a purple bar. Transcription is denoted as ‘TXN,’ and Cas9/gRNA-mediated editing as ‘Edit’