Genome editing followed by sequencing has now been used to engineer and analyse every variation of several stretches of human DNA in living cells, providing insight into the function of each constituent nucleotide molecule. See Letter p.120
You were shaped by both nature and nurture, by your DNA and your environment. How do the differences in your genome, compared with those of other people, affect who you are? In this issue, Findlay et al.1 (page 120) put a combination of human-genome editing2 and in-depth gene sequencing to brilliant use, and bring us one large step closer to a general answer to this monumental question.
Understanding our genes is a challenge. Human DNA is around 6.5 billion base pairs long — as many letters as there are in more than 5,000 copies of James Joyce's Ulysses — and differences in the DNA of any two people occur approximately every 1,000 base pairs. Although some of these differences have a large impact (for example, preventing certain adults from drinking milk), most have either a minor effect (such as making one person 0.5 centimetres taller than another) or none at all. DNA can be read quickly and inexpensively with today's technology, but understanding what any changes mean can be even harder than reading certain passages by Joyce: some genetic text might as well be written in Elvish. At the next level up, and with some noteworthy exceptions, we have little understanding of which parts of the proteins and RNA molecules encoded by genes matter to the cell and the organism, and which do not.
This seems a simple enough problem to solve. To discover whether variations in a gene matter, alter them and see what happens. However, this experiment has long proved challenging, because our cells have evolved to protect their DNA from such change on demand. The solution is gene editing2, the first step of which is to cut DNA inside the living cell. This was initially achieved using an engineered enzyme called a zinc-finger nuclease3, and more recently, two other types of enzyme — TALENs4 and CRISPR-associated enzymes5 — have also been co-opted for this purpose. Cells can repair DNA breaks by either joining the two ends back together or patching up the break using genetic information from a different DNA molecule that has an identical or similar sequence. In one form of human-gene editing6, this second pathway is subverted, by having the cell repair the break using a new piece of DNA that contains a desired mutation.
Findlay et al. built on this latter approach to address a fundamental question: which parts of a gene are actually useful? First, they focused on a gene whose function is vital for cell survival, DBR1, engineering a stretch of 75 nucleotides such that every possible single-nucleotide mutation was made individually. Genome editing has previously been used to create a small set of desired changes in a mammalian gene6,7, or to edit multiple genes in the same pathway8,9, but never before has every change possible been made (until now, experiments of such scale were achievable only in budding yeast10). The result is a group of cells that each carry a different DNA sequence, much like a set of sentences that each differ by one typographical error (Fig. 1).
To determine which changes are beneficial to the cell and which detrimental, Findlay and colleagues used deep sequencing, which reads every copy of every gene in every cell of a population. Immediately after editing, the cells are a kaleidoscope of genetic diversity. Edited cells account for only 1–3% of the total cell population (lower than seen in other studies2), but this is not a real problem because deep sequencing can identify even very rare DNA sequences.
After a few days, a stark change occurs. Many new sequences disappear or diminish in number. This is survival of the fittest at the cellular level. The authors found that cells unlucky enough to acquire a change in a nucleotide needed for gene function died immediately, but cells that had more-benign errors lived on. This experiment provides a remarkable functional map of this bit of genetic text — we know whether each and every position makes a useful contribution to the working of the whole protein.
Some genetic changes do not affect what a protein does, but rather how messenger RNA molecules are put together such that sections that do not specify the sequence of a protein are removed (a process known as splicing). Findlay and co-workers investigated how DNA sequence affects splicing in BRCA1, mutations in which cause breast cancer, in some cases because of improper splicing.
The authors generated almost every possible sequence in a 6-base-pair stretch of BRCA1, and investigated which sequences helped the gene to be copied into normal RNA, and which prevented it. They took this remarkable group of 4,048 different kinds of cell, growing side by side in the same Petri dish, and measured how often each sequence occurred in BRCA1 DNA and the corresponding RNA. Some sequences were never found in RNA, giving an insight into which genetic signals control how RNA acquires its fully functional form.
Findlay and colleagues have provided a way to find meaning in the text of human DNA, by systematically analysing each nucleotide in a gene in its normal setting in the chromosome. All you need is a robust way to edit your region of interest3,4,5 and a method to assay the cellular consequences of editing. The word 'random' often has negative connotations in science, but not in this instance. Making random changes in a gene and letting nature take its course is enormously informative. For instance, a major challenge for women who carry a mutation in BRCA1 is to determine the risk of contracting cancer for their specific mutation. Findlay and co-workers' approach can be used to address this problem and to determine which specific BRCA1 mutations are the most worrisome.
More generally, the juxtaposition of genome editing and deep-sequencing technologies will, without doubt, provide a basis for progress in our quest to understand how our DNA makes us who we are. The authors' work provides an excellent case in point to support the words of geneticist Sydney Brenner11: “Progress in science results from new technologies, new discoveries and new ideas, probably in that order.”
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The Analyst (2016)