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Generating custom human DNA sequences based on traits such as eye colour?

Generating custom human DNA sequences based on traits such as eye colour?


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I'm wondering if it would be possible to create software (unless some already exists, but I couldn't find any) to generate human DNA (the base pairs on the double helix) containing genes representing specific permutations (eye colour, hair colour, etc.)?

Basically, something like the "character builder" from those "Saints Row"-style video games, except with actual human chromosomes, enabling you to essentially create a 'custom' human.

Of course, that is assuming that all human DNA has a common structure, and that the entire sequence can either be assembled from individual chromosomes, or by using a reference genome and modifying specific genes/chromosomes according to user input. Is this the case?

One setback here, of course, could be the number of bases in each chromosome, which ranges between 100 million and 250 million, and the (approximately) 23000 human genes - a lot of data to manipulate.


A major limiting factor in such an exercise is the complexity of the biological process under question (eye colour etc.,) and thus, our nascent understanding of these. What I mean is that there is not a single gene that determines a given characteristic, rather it is the complex interaction of a set of genes in different conditions responding to environmental cues and such. We are barely at the level of scratching the surface when it comes to understanding the mechanisms that underlie such outputs. And so, we are long ways off (maybe hundreds of years?) before we can even start thinking about coming up with feasible approaches to such objectives.


What Is CRISPR?

CRISPR technology is a simple yet powerful tool for editing genomes. It allows researchers to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases and improving crops. However, its promise also raises ethical concerns.

In popular usage, "CRISPR" (pronounced "crisper") is shorthand for "CRISPR-Cas9." CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.

CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to foil attacks by viruses and other foreign bodies. They do so primarily by chopping up and destroying the DNA of a foreign invader. When these components are transferred into other, more complex, organisms, it allows for the manipulation of genes, or "editing."

Until 2017, no one really knew what this process looked like. In a paper published Nov. 10, 2017, in the journal Nature Communications, a team of researchers led by Mikihiro Shibata of Kanazawa University and Hiroshi Nishimasu of the University of Tokyo showed what it looks like when a CRISPR is in action for the very first time. [A Breathtaking New GIF Shows CRISPR Chewing Up DNA]


Family ties

In December 2017, genetic genealogist Barbara Rae-Venter got the call that would propel family-tree forensics into the public eye. She was running a business that used GEDMatch to find clients’ long-lost relatives when she heard from a California detective who had found some old DNA evidence and was trying to reopen the case of the Golden State Killer, a serial rapist and murderer who committed a string of crimes in the 1970s and 1980s.

Combining DNA samples with family trees is the core of forensic genetic genealogy. The process rests on the simple statistical rules of genetics. A parent and child, or two siblings, share 50% of their DNA. Grandparents and grandchildren share 25%. Even distant relatives share small portions of DNA. This allows consumer genetic-testing companies such as Ancestry in Lehi, Utah, and 23andMe in Sunnyvale, California, to estimate relationships between two individuals who have submitted samples, as far out as fourth cousins (who share a pair of great-great-great grandparents). Anyone can upload the results of their own DNA test to databases such as GEDMatch.

Crack down on genomic surveillance

Rae-Venter found two GEDMatch profiles that looked to be distant cousins of the suspect, and used that information to work backwards and find their great-grandparents. Then, she moved forward in time to trace their descendants, focusing on California during the time the crimes were committed. After two months, Rae-Venter handed the detective the names of three brothers. DNA from a cigarette discarded by one brother matched the sample, and on 24 April 2018, police arrested Joseph DeAngelo — in the first criminal case to be solved using the technique. (DeAngelo pleaded guilty to multiple counts of rape and murder and was sentenced to life in prison last month.)

Following DeAngelo’s arrest, forensic genetic genealogists such as Rae-Venter and CeCe Moore (who joined Parabon in May 2018) helped to solve similar rape and murder cases at a rapid clip. Although a few ethicists raised concerns about privacy, media coverage of the cases was overwhelmingly positive. “I was actually surprised there wasn’t more criticism,” says geneticist Ellen McRae Greytak, bioinformatics chief at Parabon.

And then the Utah case hit the media, and the criticism came crashing in.


How are traits passed on through DNA?

The traits of a living thing depend on the complex mixture of interacting components inside it. Proteins do much of the chemical work inside cells, so they largely determine what those traits are. But those proteins owe their existence to the DNA (deoxyribonucleic acid), so that is where we must look for the answer.

The easiest way to understand how DNA is organized is to start with its basic building blocks. DNA consists of four different sugars that interact with each other in specific ways. These four sugars are called nucleotide bases and have the names adenine ( A ), thymine ( T ), cytosine ( C ) and guanine ( G ). Think of these four bases as letters in an alphabet, the alphabet of life!

If we hook up these nucleotides into a sequence--for example, GATCATCCG --we now have a little piece of DNA, or a very short word. A much longer piece of DNA can therefore be the equivalent of different words connected to make a sentence, or gene, that describes how to build a protein. And a still longer piece of DNA could contain information about when that protein should be made. All the DNA in a cell gives us enough words and sentences to serve as a master description or blueprint for a human (or an animal, a plant, or a microorganism).

Of course, the details are a little more complicated than that! In practice, active stretches of DNA must be copied as a similar message molecule called RNA. The words in the RNA then need to be "read" to produce the proteins, which are themselves stretches of words made up of a different alphabet, the amino acid alphabet. Nobel laureates Linus Pauling, who discerned the structure of proteins, and James Watson and Francis Crick, who later deciphered the helical structure of DNA, helped us to understand this "Central Dogma" of heredity--that the DNA code turns into an RNA message that has the ability to organize 20 amino acids into a complex protein: DNA -> RNA -> Protein.

To understand how this all comes together, consider the trait for blue eyes. DNA for a blue-eyes gene is copied as a blue-eyes RNA message. That message is then translated into the blue protein pigments found in the cells of the eye. For every trait we have--eye color, skin color and so on--there is a gene or group of genes that controls the trait by producing first the message and then the protein. Sperm cells and eggs cells are specialized to carry DNA in such a way that, at fertilization, a new individual with traits from both its mother and father is created.


Discussion

Our analysis indicates that positive selection on pigmentation variants associated with depigmented hair, skin, and eyes was still ongoing after the time period represented by our archaeological population, 6,500–4,000 y ago. This finding suggests that either the selection pressures that initiated the selective sweep during the Late Pleistocene or early Holocene were still operative or that a new selective environment had arisen in which depigmentation was favored for a different reason.

The high selection coefficients estimated for pigmentation genes HERC2, SLC45A2, and TYR are best understood in the context of estimates obtained for other recently selected loci. Using spatially explicit simulation and approximate Bayesian computation, selection on the LCT -13,910*T allele—which is strongly associated with lactase persistence in Europeans and southern Asians—was inferred to fall in the range 0.0259–0.0795 and to have begun around 7,500 y ago in the region between the Balkans and central Europe (37). However, another simulation-based study incorporating latitudinal effects on selection resulted in a lower estimate of S (0.008–0.018) (38). The selective advantage of the G6PD A− and Med deficiency alleles conferring resistance to malaria have been estimated at 0.019–0.048 and 0.014–0.049, respectively, in regions where malaria is endemic (39). These alleles are estimated to have arisen ∼6,357 y ago (G6PD A−) and 3,330 y ago (G6PD Med) (39). Thus, the estimates of S for the three pigmentation genes examined in this study are comparable to those for the most strongly selected loci in the human genome.

Although these estimated selection coefficients are high, they are comparable to previous estimates for genes in the pigmentation complex. The selective sweeps favoring the SLC45A2 derived allele, as well as the derived alleles of SNPs in SLC24A5 and TYRP1, which are also implicated in the lightening of skin pigmentation, are estimated to have begun between 11,000 and 19,000 y ago, after the separation of the ancestors of modern Europeans and East Asians (the ages of the selective sweeps affecting HERC2 and TYR have not yet been estimated) (14, 40). Beleza et al. (14) recently estimated the coefficient of selection at the SLC45A2 locus to be 0.05 under a dominant model of inheritance and 0.04 under an additive model. Selection favoring the derived alleles of SNPs in SLC24A5 and TYRP1 was found to be similarly strong.

Estimating selection coefficients using the ancient DNA-based simulation approach presented here offers considerable advantages over traditional methods based on allele age and frequency estimates (1): Selection coefficients are estimated over a defined period selection acting on standing variation can be accommodated and our approach is insensitive to the frequently unaccounted for uncertainties associated with allele age estimation using molecular or recombination clocks. This latter advantage is likely to result in considerable improvements in precision. However, our approach does require the assumption of population continuity and will not provide direct estimates of when a selective sweep began.

Although the strength of the selection coefficients in a certain time window can be estimated with improved precision using our ancient DNA-based simulation approach, the actual nature of the selection pressure remains unknown. However, temporal and geographical information from the prehistoric skeletal population under study can help in formulating reasonable hypotheses. Geographic variation in many functional skin pigmentation gene polymorphisms (13), and lighter skin pigmentation more generally, correlate strongly with distance from the equator in long-established populations, suggesting that selective pressure also occurred along a latitudinal gradient. The samples in our study were from between 42°N and 54°N, a latitudinal belt in which yearly average UVR is insufficient for vitamin D3 photosynthesis in highly melanized skin (4, 41). Constraints on the ability to photosynthesize vitamin D3 imposed by low incident UVR intensity may have provided significant selective pressure favoring lighter pigmentation populations in high-latitude regions such as the northern Pontic steppe belt. The need to admit UVB radiation to catalyze the synthesis of vitamin D3, together with the decreased danger of folate photolysis at higher latitudes, may account for the observed skin depigmentation from prehistoric to modern times in this region (5).

Dietary change during the Neolithization process may have reinforced selection pressure favoring depigmented skin. The individuals analyzed in this study lived ∼500–2,000 y after the arrival of farming in the region north of the Black Sea (42, 43). In many parts of Europe, the Mesolithic–Neolithic transition is associated with a switch from a vitamin D-rich aquatic or game-based hunter–gatherer diet (44) to a vitamin D-poor agriculturalist diet. In low-UV regimes such as the one prevailing in our study region, it is difficult to meet vitamin D requirements without the consumption of significant quantities of oily fish or animal liver (45, 46). The vitamin D recommended dietary allowance of 800–1,000 IU for adults requires the daily consumption of the equivalent of 100 g of wild salmon (the dietary input with the greatest measured vitamin D concentration). Isotopic evidence suggests that the populations sampled in our study continued to access aquatic resources, primarily river fish, in the Neolithic, Eneolithic, and Bronze Age, although there was considerable heterogeneity in fish consumption within the study region (47 ⇓ ⇓ –50). However, any diminution in fish consumption may have been sufficient to generate additional selective pressure favoring depigmentation at this low-incident-UVR latitude.

Although ecological and environmental factors may be sufficient to explain the observed change in European skin pigmentation, these explanations are unlikely to hold for eye and hair color. The geographic distribution of iris and hair pigmentation variation does not conform as well to a latitudinal cline model, with much of the observed phenotypic variation restricted to Europe and closely related neighboring populations (51, 52). The blue iris phenotype characteristic of the HERC2 rs12913832 G allele, for example, is almost completely restricted to western Eurasia and some adjacent regions, its descendant populations, and populations containing European admixture (51, 52). It is possible that depigmented irises or the various human hair color morphs in Europeans are by-products of selection on skin pigmentation. There is evidence for gene–gene interaction within the polygenic system governing complex pigmentation traits interactions between HERC2, OCA2, and MC1R, in particular, have been found to have a statistically significant effect on hair, iris, and skin color (36). There is also evidence for epistatic interactions between components of the melanin synthesis pathway in other mammalian model systems, including interactions between the products of ASIP, MC1R, and TYR (53). Additionally, many pigmentation genes, including TYR, HERC2, and SLC45A2 have pleiotropic effects on skin, hair, and eye color (11, 36).

Given that intraspecific pigmentation variability in other taxa, particularly avians, has been attributed to signaling and other factors associated with mate choice (54) it is possible that depigmented irises and the various hair colors observed in Europeans arose through sexual selection (7). Frequency-dependent sexual selection in favor of rare variants has been observed in vertebrates (55, 56), and such selection favoring rare pigmentation morphs could have driven alleles associated with lighter hair and eye colors to higher frequency. Once lighter hair and eye pigmentation phenotypes reached appreciable frequencies in European populations, these novel traits may have continued to be preferred as indicators of group membership, facilitating assortative mating. Assortative mating based on coloration is common in vertebrates (57), and skin pigmentation has been observed as a criterion for endogamy in modern human populations (58, 59). In addition, there is some evidence that lighter iris colors, because of their recessive mode of inheritance, may be preferred by males in assortative mating regimes to improve paternity confidence (60). Consistent with positive assortative mating, an exact test of Hardy–Weinberg equilibrium reveals an excess of HERC2 rs12913832 homozygotes in both the modern (P = 0.0543) and ancient (P = 0.0084) East European samples genotyped here (Table S3), despite the relatively small sample sizes.

The observed excess of HERC2 rs12913832 homozygotes in the ancient sample might be explained by population stratification in a temporally heterogeneous population sample. Although we do not observe any chronological or spatial patterning of the pigmentation markers in our prehistoric sample, we cannot exclude population stratification in the absence of additional neutral SNPs. However, we note that neither the TYR nor the SLC45A2 SNPs investigated here, nor three additional SNPs investigated in the same ancient and modern samples, showed any significant observable excess of homozygotes (Table S3), suggesting that the excess of HERC2 rs12913832 homozygotes is less likely to be due to population stratification.

In sum, a combination of selective pressures associated with living in northern latitudes, the adoption of an agriculturalist diet, and assortative mating may sufficiently explain the observed change from a darker phenotype during the Eneolithic/Early Bronze age to a generally lighter one in modern Eastern Europeans, although other selective factors cannot be discounted. The selection coefficients inferred directly from serially sampled data at these pigmentation loci range from 2 to 10% and are among the strongest signals of recent selection in humans.


Body fluid identification

The ability to identify the presence of a specific body-fluid can be extremely valuable to an investigation, providing crucial information on the activities involved in an incident, particularly if it means that a DNA profile can be linked to a specific biological source. All of the presumptive/confirmatory tests currently used to identify some (but not all e.g. vaginal material, menstrual blood) body fluids have limitations, including a lack of sensitivity and specificity, and a requirement to carry out multiple tests that destroy limited samples [52]. This has led to interest in the analysis of RNA in body fluid stains, particularly given RNA can be co-extracted with DNA, allowing parallel production of a DNA profile alongside body fluid testing [53].

Identification of body fluids using RNA profiling is based on the principle that although DNA content is the same in most cell types, RNA differs depending on cell type and function. The production of RNA is therefore tissue-specific, such that each body fluid has a specific gene expression pattern. The presence of tissue-specific RNA types in a sample can therefore indicate the presence of specific body fluids [54]. Research in this area has focused on large-scale screens for differentially expressed RNAs followed by the development of PCR-based assays to target individual or small numbers of markers. Many of these assays employ reverse transcription endpoint (RT-PCR) or quantitative real-time (RT-qPCR) PCR [55], which have the benefit of compatibility with existing technologies in forensic laboratories, although increasingly studies are utilising the power of MPS for identification and analysis of tissue-specific RNAs [56–59].

Initial assays focused on identifying body fluid-specific messenger RNA (mRNA) markers, and development of multiplexes indicating the presence of single or multiple body fluid types, the latter of which is particularly useful when analysing mixed samples (reviewed in [55,60]). However, the susceptibility of mRNAs to degradation has limited their application to forensic samples, and mRNA assays also suffer from limitations including variation in sensitivity and specificity and interpretational challenges [61–64]. More recently, focus has been on micro RNAs (miRNAs) as alternative markers for body fluid identification [65]. Many of these regulatory RNAs, which target mRNAs for degradation or silencing, also show tissue-specific expression, and have the benefit of increased stability compared with mRNA as a result of their smaller size and incorporation into a protein complex within the cell [66]. A variety of miRNAs have been identified as potential markers for forensically relevant body fluids, and although it is unlikely any miRNAs are specific to single body fluid types, a number of assays have been developed that incorporate panels of multiple differentially expressed miRNAs that appear to identify specific body fluids [67–73]. Although miRNA-based assays suffer from some of the same challenges as mRNA-based assays, particularly in terms of interpretation, miRNAs have great potential as body fluid markers [67,68,70]. Further study to identify the best sets of miRNAs to unambiguously identify different body fluids and detailed validation of the resulting assays may give the forensic community a reliable test for the identification of body fluids [65]. Micro RNA markers also hold promise for other forensic applications, including estimating the time of deposition of body fluid stains [74,75] and the post-mortem interval [76].


Crispr: is it a good idea to ‘upgrade’ our DNA?

L ast year Tony Perry made mice that would have been brown-furred grow up white instead. That Perry, a molecular embryologist at the University of Bath, tweaked their coat colour isn’t new – scientists have been making so-called knock-out mice, in which certain genes are disabled, since the technique was invented in 1989. It is a long and cumbersome procedure that involves combining pieces of DNA in embryonic stem cells and mouse breeding.

But Perry, who published his study in December, didn’t use this method. Instead he used a new genome-editing technology that has been taking the scientific world by storm since it was first developed from the bacterial immune system in 2012, and shown to work in human cells in 2013.

The powerful tool, known as Crispr, allows the precise and easy manipulation of the DNA in the nucleus of any cell. Make the manipulations in sperm, egg or a one-cell embryo, which is just about to start replicating its DNA, and they can become permanently sealed in the so-called germ line, to be inherited by future generations. Using the procedure on the germ line, Perry inactivated a key gene for mouse coat colour.

But Perry’s work added a unique flourish. He did the editing not in a one-cell mouse embryo – which is how most animal germ-line editing by Crispr has been done to date – but earlier, during the process of fertilisation, by injecting the Crispr components and the mouse sperm into the mouse egg at the same time. It is the same technique – intracytoplasmic sperm injection (ICSI) – widely used in IVF. And it worked. “This or analogous approaches may one day enable human genome targeting or editing during very early development,” notes the paper published in the journal Scientific Reports. If human germ-line editing were ever to be used clinically, incorporating Crispr into the ICSI phase of IVF is how it might be.

That prospect tantalises Perry because it raises the possibility of generating offspring that carry either no risk or a reduced risk of some genetic diseases. Perry suggests it might one day be possible to correct a harmful mutation in the BRCA1 gene and stop someone inheriting that predisposition to breast cancer. “You will be able to eradicate it from your descendants,” he says.

Crispr can be thought of as a pair of molecular scissors guided by a satnav. The scissors are a DNA-cutting enzyme they snip at a precise point in the cell’s DNA specified by researchers using a customised guide molecule, a single short piece of RNA, DNA’s chemical cousin. The DNA-cutting enzyme is known as Cas9, hence the technique is often written Crispr-Cas9.

The genome editing occurs as the cell rushes to naturally repair the break made by the scissors. The cell’s repair often isn’t exact enough for the gene that has been cut to keep working and the gene is effectively knocked out or turned off. More complex to accomplish, though more precise, genes can also be corrected or whole new genes added if a new piece of DNA is included along with the Crispr machinery. It becomes patched in during the cellular repair process.

Germ-line genome editing is highly controversial, even for medical purposes. Since the development of genetic engineering in the 70s there has been a “fairly undisturbed” consensus that human germ-line genetic modification – with the worries it raises about “playing God” and “designer babies” – is off bounds, says Peter Mills, assistant director of the UK Nuffield Council on Bioethics and the council’s lead on genome editing. According to Unesco’s Universal Declaration on the Human Genome and Human Rights, germ-line interventions “could be contrary to human dignity”.

The UK government’s decision this February to allow mitochondrial substitution in the clinic to prevent embryos developing with mitochondrial diseases, a form of germ-line therapy, was premised on the basis that the small amount of DNA mitochondria contain is found outside the cell nucleus. There is no modification to the DNA in the nucleus, the real stuff that makes us who we are.

Crispr co-developer Jennifer Doudna.

But there has also never before been a tool shown to be sufficiently effective or reliable enough to seriously consider conducting human genetic engineering. Other precise genome editing tools – Zinc Finger Nuclease (ZFN) and Talen – have been around longer but Crispr is much easier and cheaper to use, accelerating the science and potential applications. “The revolution is in access,” says Dana Carroll, a biochemist at the University of Utah who works on improving the tools. “The technology is developing very rapidly.”

The potential for modifying humans weighs on the minds of some scientists, particularly since the publication last month of a Chinese paper which reported using Crispr to genome-edit human embryos for the first time. (The aim was to correct the gene defect that causes the blood disorder beta-thalassaemia and non-viable embryos, which couldn’t have resulted in live birth, were used.)

In an opinion piece published in the journal Science in March, a group of US scientists led by the Crispr co-developer Jennifer Doudna from the University of California, Berkeley recommended steps be taken to “strongly discourage” any attempts at germ-line modification therapy that would produce genome-edited humans while the social and ethical implications are considered. They are calling for an international meeting to consider the appropriate way forward for use. “Lets do this now before the technology is applied in ways which people might feel very uncomfortable about,” says Doudna.

A second group writing in the journal Nature went further, suggesting a moratorium on research where human germ cells are edited for fear of where it might lead. The backlash should a modified human be born could, they warn, harm work to develop genome-editing therapy for adults and children where the modifications aren’t passed on. “People might not prise apart the nuances,” says Edward Lanphier, the president and CEO of California-based Sangamo Biosciences, which is pursuing that work.

Indeed, using Crispr in non-reproductive cells – somatic cells – could cure many diseases. Researchers believe it is likely to make it to the clinic sooner. Those modifications can be permanent to the individual’s cells in so far as those cells multiply or remain alive a long time, but they aren’t inherited by future generations. “It has none of the ethical burdens these steps towards germ-line modification would be shouldering,” says Dana Carroll.

The downside is that using Crispr on somatic cells is far more complex: humans have trillions of cells and many different cell types. The genome-tinkering machinery has to be delivered to a sufficient proportion of the specific problem cells to bring about a therapeutic effect.

Thus far, Crispr is too new for any somatic cell editing therapy to have entered clinical trials. But Jennifer Doudna expects them to begin within a few years. “If they go well, I think we could see approved therapeutics within a decade,” she says. In the lab meanwhile, using both human somatic cell cultures and animal models, researchers are experimenting with treating diseases including sickle cell anaemia, severe combined immunodeficiency (SCID), beta-thalassemia, haemophilia, muscular dystrophy and cystic fibrosis. (The approach for these single gene disorders is based on adding a new piece of DNA to correct the faulty gene.)

New companies – Editas Medicine, Crispr Therapeutics and Intellia Therapeutics – have sprung up, licensing Crispr technology to develop somatic cell editing for the clinic. None have yet announced which diseases they are pursuing and a legal battle around who exactly owns the rights to Crispr technology rages. But Intellia has a deal with pharmaceutical giant Novartis to focus on cancer and genetic disorders arising in blood cells.

Somatic cell editing is a sort of upgrade to an earlier technique for curing single gene disorders known as gene therapy. That was first tried in the 90s and hit setbacks. But in 2012 the first gene therapy in Europe – to treat a disease in which a person lacks a protein needed to break down fat molecules – was approved.

Gene therapy introduces a whole new working copy of a gene, which randomly incorporates into the genome to do the job of the faulty one. Genome editing is different in that it precisely targets the existing faulty gene for knock-out or correction. That means the gene’s setting doesn’t change, so doctors neither have to worry that it will incorporate somewhere that causes other genes to be inadvertently turned on, nor that the gene won’t work as normal, for example by not producing the right amount of protein.

“You are keeping everything natural,” says Jennifer Puck, a SCID expert at the University of California, San Francisco, who is exploring the potential of Crispr to treat the disease. Notoriously, in an early trial to treat SCID using gene therapy, the replacement gene incorporated in an unfortunate location and caused leukaemia in five of the 20 patients treated. That problem has been fixed, but the technique can still be tricky to get right.

Human embryos in the lab. Photograph: Bloomberg via Getty Images

One challenge shared by Crispr and gene therapy is how to get the gene – or Crispr machinery – inside cells. Methods being adopted from gene therapy to encapsulate and deliver it range from modified viruses to nanoparticles. All are still far from perfect. “People are working hard on delivery and it is problem that will be solved,” says Doudna. “But it is not solved today.”

Delivery is made easier, however, when the cells can be removed for editing. Once outside the body they can be purified, expanded in culture, and checked via genome sequencing to ensure the editing has been successful. That means the early clinical impact of Crispr is likely to be in treating genetic diseases arising in blood cells such as sickle cell anaemia, SCID and beta thalassemia. Doctors are adept at extracting blood and bone marrow (rich in blood stem cells, which give rise to all other blood cells), isolating particular cells for manipulation, and then re-implanting them. Diseases where the cells can’t be removed for treatment will require more work. That includes haemophilia, muscular dystrophy, and cystic fibrosis, which predominantly arise in the liver, muscle and lung cells respectively.

A sense of what Crispr’s future might hold can be seen in the progress of Sangamo Biosciences, which is clinically trialling a somatic cell editing therapy for HIV with efficacy data anticipated by the end of the year. “The goal is to have a functional cure for HIV,” says Edward Lanphier. That would let a patient stop taking antiretroviral drugs for a number of years.

The trial uses the older ZFN technology, for which the company holds the intellectual property rights. The aim, based on replicating a rare naturally occurring mutation that makes some people resistant to HIV infection, is to turn off the CCR5 gene in patients’ T-cells – a type of white blood cell important in the immune system. That gene produces a protein that HIV uses to enter those cells and cause infection.

The ZFNs are put to work outside the body on cells extracted from the patient’s blood. The edited cells are returned in the hope of creating a population of HIV-resistant T-cells that can fight the virus. Just like with Crispr, the custom-designed ZFNs, which come as a pair where a protein structure containing zinc molecules does the guiding, cut the DNA in a precise location – the CCR5 gene – before the cell’s natural but inaccurate repair process kicks in, knocking out the gene. “It really illustrates the power of the approach for treating disease,” says Jennifer Doudna.

Meanwhile, whether editing the human germ line has any clinical future will depend on being able to do it accurately and safely enough, and also whether society finds it acceptable. The conclusion of the Chinese study was that there are still significant technical challenges to be solved before clinical application of Crispr for editing embryos is possible. Problems include “off-target” effects, where the DNA is also snipped at places it shouldn’t be, and “genetic mosaicism”, where the result has a mixture of modified and unmodified cells. Nonetheless, many believe things will improve with more research. It is not safe enough yet, says Robin Lovell-Badge, a researcher who studies germ-cell biology at the Francis Crick Institute, but “it is going to get there fairly soon”.

What specific therapeutic uses might be envisaged? For Lovell-Badge there aren’t many. Pre-implantation genetic diagnosis (PGD) – embryo screening to select those that don’t carry certain disease risks – is already in use for a long list of genetic conditions including the BRCA1 gene, he points out. But he concedes Crispr could have a place when multiple embryos are affected, such as in the case of men who are infertile or sub-fertile because of mutations on the Y chromosome. Any male embryos (produced by IVF) will also have that same mutation.

However, others see wider use. Unlike embryo screening, germ-line editing would not require multiple embryos, which some couples don’t have. It could deal easily with multiple genetic conditions where finding unaffected embryos is a challenge. And it wouldn’t involve discarding any embryos, which some people feel uncomfortable with. “There is an argument for genome engineering in embryos to repair genes that clearly predispose to disease,” says Perry.

Some people may even think it important to use Crispr to make better humans, not just preemptively stamp out disease. There are gene variants which confer extra-strong bones, low Alzheimer’s risk or viral resistance such as to HIV. Beyond that, what about enhancements such as living longer, improved cognition, or altered physical attributes? “I am sure there are broader human characteristics that people would like to be able to modulate,” says Dana Carroll. But he adds that presently those kind of multi-genetic traits would be difficult to edit in because we don’t fully understand their basis, let alone what unintended consequences might result.

The UK has a cautiously progressive regulatory system that would apply to developments in human germ-line editing. Any research on germ cells needs to be licensed by the Human Fertilisation and Embryology Authority (HFEA). Parliamentary approval would be needed for therapeutic use. According to a list of research projects using human embryos currently being carried out in the UK provided by the HFEA, none appear to involve genome editing. But Lovell-Badge says he is aware of “several groups” in Britain interested in using it to “answer some basic research questions”. (Meanwhile the main public funder of research in the US - the NIH - says it won’t fund any use of gene-editing technologies in human embryos.)

It is possible the consensus against modifying humans could change, says Peter Mills, adding that the Nuffield Council is planning a report to consider the ethics of human germ-line editing. There was a new social understanding reached in the UK with the mitochondrial decision a move from simply selecting human embryos to modifying them. “You can treat [nuclear DNA] as a limit that you don’t cross or a threshold that you very carefully step over,” he says.

If it can be done accurately and safely, human germ-line therapy is a possibility Doudna says she is certainly open to. “People get comfortable with technologies,” she says, citing how over time society has become relaxed about the use of IVF. “I suspect this will be the same.”


Acknowledgements

We are indebted to the owners of the gourd for allowing access to the blood sample inside it and to John Novembre for advice and assistance with the ancestry analysis. This work is supported by FEDER and Spanish Government grants BFU2012-38236 and the Spanish Multiple Sclerosis Netowrk (REEM) of the Instituto de Salud Carlos III (RD12/0032/0011) to A.N., BFU2011-28549 and ERC Starting Grant (260372) to T.M.-B. and BFU2012-34157 to C.L.-F. and S.C. and a predoctoral fellowship from the Basque Government (DEUI) to I.O.


MATERIALS AND METHODS

Specimens: Specimens for resequencing were obtained from the Coriell Institute in Camden, New Jersey. Specimens for genotyping were of self-reported European descent, of different age, sex, hair, iris, and skin shades and they were collected using informed consent guidelines under Investigational Review Board guidance. Donors checked a box for blue, green, hazel, brown, black, or unknown/not clear iris colors, and each had the opportunity to identify whether iris color had changed over the course of their lives or whether the color of each iris was different. Individuals for whom iris color was ambiguous or had changed over the course of life were eliminated from the analysis. In addition, for 103 of the subjects, iris colors were reported using a number from 1 to 11 as well, where 1 is the darkest brown/black and 11 is the lightest blue, identified using a color placard. For these subjects, we obtained digital photographs of the right iris, where subjects peered into a box at one end at the camera at the other end to standardize lighting conditions and distance and from which a judge assigned the sample to a color group. Comparing the results of the two methods of classification, 86 of the classifications matched. Of the 17 that did not, 6 were brown/hazel, 7 were green/hazel, and 4 were blue/green discrepancies although none were gross discrepancies such as brown/green, brown/blue, or hazel/blue. Although such an error is tolerable for identifying sequences marginally associated with iris colors, the use of the sequences described herein for iris color classification would therefore likely require digitally quantified iris colors (which we have begun to accumulate and will present elsewhere).

SNP discovery: We obtained candidate SNPs from the National Center for Biotechnology Information (NCBI) Single Nucleotide Polymorphism Database (dbSNP), which generally provided more candidate SNPs than were possible to genotype. We focused on human pigmentation and xenobiotic metabolism genes, selected on the basis of their gene identities, not their chromosomal position. For some genes, the number of SNPs in the database was low and/or some of the SNPs were strongly associated with iris colors, warranting a deeper investigation. For these genes we performed resequencing and of the genes discussed in this article, 113 SNPs were discovered in CYP1A2 (7 gene regions, 5 amplicons, 10 SNPs found), CYP2C8 (9 gene regions, 8 amplicons, 15 SNPs found), CYP2C9 (9 gene regions, 8 amplicons, 24 SNPs found), OCA2 (16 gene regions, 15 amplicons, 40 SNPs found), TYR (5 gene regions, 5 amplicons, 10 SNPs found), and TYRP1 (7 gene regions, 6 amplicons, 14 SNPs found). Resequencing for these genes was performed by amplifying the proximal promoter (average 700 bp upstream of transcription start site), each exon (average size 1400 bp), the 5′ and 3′ ends of each intron (including the intron-exon junctions, average size ∼100 bp), and 3′ untranslated region (UTR average size 700 bp) sequences from a multi-ethnic panel of 672 individuals (450 individuals from the Coriell Institute's DNA Polymorphism Discovery Resource, 96 additional European Americans, 96 African Americans, 10 Pacific Islanders, 10 Japanese, and 10 Chinese these 672 individuals represented a set of samples separate from that used for the association study described herein). PCR amplification was accomplished using pfu Turbo polymerase according to the manufacturer's guidelines (Stratagene, La Jolla, CA). We developed a program (T. F rudakis , M. T homas , Z. G askin , K. V enkateswarlu , K. S uresh C handra , S. G injupalli , S. G unturi , S. N atrajan , V. K. P onnuswamy and K. N. P onnuswamy , unpublished results) to design resequencing primers in a manner respectful of homologous sequences in the genome, to ensure that we did not coamplify pseudogenes or amplify from within repeats. BLAST searches confirmed the specificity of all primers used. Amplification products were subcloned into the pTOPO (Invitrogen, San Diego) sequencing vector and 96 insert-positive colonies were grown for plasmid DNA isolation (the use of 670 individuals for the amplification step reduced the likelihood of an individual contributing more than once to this subset of 96 selected). We sequenced with an ABI3700 using PE Applied Biosystems BDT chemistry and we deposited the sequences into a commercial relational database system (iFINCH, Geospiza, Seattle). PHRED-qualified sequences were imported into the CLUSTAL X alignment program and the output of this was used with a second program that we developed (T. F rudakis , M. T homas , Z. G askin , K. V enkateswarlu , K. S uresh Chandra , S. G injupalli , S. G unturi , S. N atrajan , V. K. P onnuswamy and K. N. P onnuswamy , unpublished results) to identify quality-validated discrepancies between sequences. We selected those for which at least two instances of PHRED identified variants that scored ≥24, and each of these SNPs discovered through resequencing were used for genotyping.

Genotyping: For most of the SNPs, a first round of PCR was performed on the samples using the high-fidelity DNA polymerase pfu Turbo and the appropriate resequencing primers. Representatives of the resulting PCR products were checked on an agarose gel, and first-round PCR product was diluted and then used as template for a second round of PCR. The two rounds were necessary due to the fact that many of the genes we queried were members of gene families, the SNPs resided in regions of sequence homology, and our genotyping platform required short (∼100 bp) amplicons. For those remaining, only a single round of PCR was performed. Genotyping was performed for individual DNA specimens using a single base primer extension protocol and an SNPstream 25K/ultra-high throughput (UHT) instrument (Beckman Coulter, Fullerton, CA, and Orchid Biosystems, Princeton, NJ). Genotypes were subject to several quality controls: two scientists independently pass/fail inspected the calls, requiring an overall UHT signal intensity >1000 for >95% of genotypes and clear signal differential between the averages for each genotype class (i.e., clear genotype clustering in two-dimensional space using the UHT analysis software).

Statistical methods: To test the departures from independence in allelic state within and between loci, we used the exact test, described in Z aykin et al. (1995). Haplotypes were inferred using the S tephens et al. (2001) haplotype reconstruction method. To determine the extent to which extant iris color variation could be explained by various models, we calculated R 2 values for SNPs, haplotypes, and multilocus genotype data by first assigning the phenotypic value for blue eye color as 1, green eye color as 2, hazel eye color as 3, and brown eye color as 4. Biogeographical ancestry admixture proportions were determined using the methods of H anis et al. (1986) and S hriver et al. (2003) within the context of a software program we developed for this purpose, which will be presented elsewhere (T. F rudakis , Z. G askin , M. T homas , V. P onnuswamy , K. V enkateswarlu , S. G unjupulli , C. B onilla , E. P arra and M. S hriver , personal communication). For R 2 computation, we used the following function: Adj-R 2 = 1 – [n/(np)](1 – R 2 ), where n is the model degrees of freedom and np is the error degrees of freedom. To correct for multiple tests, we used the empirical Bayes adjustments for multiple results method described by S teenland et al. (2000). Linkage disequilibrium (LD) for pairs of SNPs within a gene was determined using the Zaykin exact test and a cutoff value of |D′| ≥ 0.05 (P value < 0.05 Z aykin et al. 1995).


Results

Cho codes for a subunit of vesicular ATPase

cho is an X-linked recessive eye color mutation that is also associated with brown pigmentation in the Malpighian tubules. It was originally described by Sturtevant (Lindsley and Zimm 1968 Sturtevant 1955). Both the amounts of ommochromes and pteridines present in cho eyes are decreased compared to wild type (Ferre et al. 1986 Reaume et al. 1991). The cytological position of cho is 3F1� and Sturtevant placed it close to echinus (ec). Three overlapping deficiencies, Df(1)BSC834, Df(1)ED6716, and Df(1)BSC877, which together removed regions around and including ec, were used for the first deletion mapping ( Table 2 ). Only heterozygotes for cho and Df(1)ED6716 showed the cho phenotype, indicating that cho was in the region removed exclusively by Df(1)ED 6716 ( Table 2 ). Genomic deficiencies were made using the Flp-FRT method (Parks et al. 2004). Deletion mapping with these identified a 47.8-Kbp region that contained the cho gene. Within it were three candidate genes: VhaAC39-1, a subunit of vacuolar ATPase CG42541, a member of the Ras GTPase family and CG15239, which has an unknown function ( Table 2 ).


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