High-throughput interrogation of genome function and cellular lineage

Abstract

Mutations can reveal how biological functions are encoded in our DNA, and how biological specimens relate to one another. In nature, mutations occur infrequently and are subject to natural selection. Therefore, to better learn how the DNA sequences within genomes function, methods to deliberately create mutations and study their effects have been developed and employed broadly. Recently engineered genome editing technologies constitute a means of inducing mutations at a high frequency and in a targeted fashion, allowing researchers to effectively rewrite the DNA code of a living cell’s genome. One such technology called CRISPR/Cas9, has enabled genome editing at unprecedented ease and scale. Here, I describe implementations of CRISPR/Cas9 genome editing to generate high allelic diversity at targeted loci. Experimental quantification of genome editing outcomes via next-generation sequencing is used to investigate two basic biological questions: 1.) How mutations impact the function of genomic sequences, both coding and regulatory, and 2.) How cells in the body relate to one another by way of a developmental lineage. We investigated how mutations impact the function of DNA in two ways. First, we established and optimized a CRISPR/Cas9-mediated method to introduce all possible single nucleotide variants over a genomic region to determine the effects of each one in parallel. We employ this method, called ‘saturation genome editing’, to investigate thousands of variants in BRCA1, a gene in which loss-of-function variants cause hereditary breast and ovarian cancer predisposition. The high accuracy of the data suggests this will be a powerful method for interpreting variants encountered clinically. Second, to probe vast expanses of genomic sequence for functional effects on gene regulation, we devised a method to introduce and assay thousands of large deletions in a high throughput manner. For one gene, HPRT1, we use this method to show that distal regulatory elements are unlikely to be required for the gene’s expression. We anticipate these two methods will be powerful and complementary tools for identifying critical regions of the genome and dissecting how they function. Towards understanding how an entire organism develops from a single fertilized egg, we developed an approach to record relationships between individual cells. We use CRISPR/Cas9 to create diverse mutations in a short DNA barcode present within each cell of a growing organism, such that the ancestral relationship between two cells can be determined by how similar the cells’ barcodes are to one another. Determining the barcode sequences of hundreds of thousands of cells sampled from grown organisms allows us to construct lineage trees that reveal how sequential cell divisions give rise first to embryonic germ layers and then to the cell types, tissues and organs of fully formed organisms. Future use of this method, which we call ‘GESTALT’, will elucidate cell lineage in multicellular systems for normal development and disease. Potential improvements and applications of these methods are described in a concluding section.