CRISPR Human Babies: Too soon, not never

Guest post by Thomas Clements

Jiankui He made waves in November 2018 when he announced he had successfully genetically modified human twin girls using CRISPR. How did he do it? What are the ethical concerns? Are the others out there? Is this a science fiction horror movie come to life? How should we move forward as a community?

CRISPR Background

When scientists talk about gene editing, they are specifically speaking about tools that target and modify the DNA. Because they target DNA, these tools are used to create novel genetic mutant lines and their induced changes are not transient. DNA is composed is four individual bases known as nucleotides (denoted A, C, T, and G). These nucleotides bind with each other and then wrap around to form a double helical structure as seen below.

Editing DNA is initiated by inducing novel double-strand breaks (DSBs) in the DNA itself, which are commonly initiated through three methods: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the recently developed the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) endonuclease mechanism.

Once a DSB break occurs, cells respond through two competing mechanisms: the dominant non-homologous end-joining (NHEJ) and homologous recombination (HR) (Huertas et al., 2008). This dominant NHEJ process is error-prone and leads to insertions and/or deletions (indels) of nucleotides at the break site. These indels can then knockout (KO) gene function by either removing necessary nucleotide base pairs (bps) or creating frame-shift mutations (protein codons are read in groups of 3, so a mutation that is not a multiple of 3 results in a disruption of the protein sequence downstream the mutation site). The CRISPR-Cas9 gene editing system is the simplest reverse genetic tool available, but it only produces indels on the order 10 bps on average (Carrington et al., 2015).

CRISPR History

The clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) endonuclease system is present in roughly 40% of sequenced bacteria and 90% of sequenced archaea (Wei et al., 2013). In bacteria and archaea, CRISPRs serve as an adaptive immune system. When a foreign nucleic acid invades a host cell and it is degraded, a small portion of it is incorporated into the CRISPR array in the form of a small spacer. This locus can then produce small RNAs that bind and guide Cas endonucleases to cleave previously degraded foreign nucleic acids (Wei et al., 2013). This mechanism was first observed in 1987 by Japanese researchers while they were researching the sequence that encodes for alkaline phosphatase in bacteria (Ishino et al., 1987).

The type II CRISPR system was adapted for gene editing purposes because of its simplicity: it uses only a single endonuclease (Cas9) and two RNA components: crispr RNA (crRNA), which contains the 20 bp target site and transactivating crRNA (tracrRNA), which is required for processing (Gonzales and Yeh, 2014). In terms of gene editing, the crRNA and the tracrRNA have been combined into a single guide RNA (sgRNA). Using this sgRNA to cleave a novel DNA site of interest was first shown in Virginijus Siksnys’ lab (Gasiunas et al., 2012) and shortly afterwards in a collaboration by Emmanuelle Charpentier and Jennifer Doudna (Jinek et al., 2012). However, genome editing at a novel site in human and mouse cells using CRISPR was first shown in Feng Zhang’s lab (Cong et al., 2013) and shortly afterwards in George Church’s lab (Mali et al., 2013).

CRISPR Simplicity

The type II CRISPR/Cas9 mechanism from S. pyogenes has been altered to use synthetic single guide RNAs (sgRNAs) that direct site-specific cleavage of a target genomic DNA sequence in vivo through the activity of the Cas9 endonuclease (Chang et al., 2013; Hwang et al., 2013; Wei et al., 2013). The sgRNA first binds to the Cas9 endonuclease and then leads it to the gene of interest through complementary base pair binding to the target site (Gasiunas et al., 2012). By using in vitro transcribed Cas9 and an sgRNA for a specific gene, a novel DSB can be created in genes of interest, which then results in insertions and deletions (indels) from errors in NHEJ that can knockout genes by inducing frame shift mutations.

Initial targeting of genes in zebrafish using the CRISPR-Cas9 system

My specialty is the use of zebrafish in genetic research. Zebrafish are a fantastic model organism because it has many biological features that make it advantageous in the scientific setting compared to the mouse. Zebrafish, Danio rerio, are less expensive to care for, reproduce at a higher frequency as well as number, and fertilization is external reducing the effort required for genetic manipulation in early development. The resulting embryos are transparent, thus allowing for visual observation throughout development (Kimmel et al., 1995).

CRISPR targeting in zebrafish was first reported by Keith Joung’s lab (Hwang et al., 2013), in which researchers used a novel sgRNA containing more tracrRNA-derived sequences at the 3′ end than a previously reported in vitro counterpart (Jinek et al., 2012). Here, researchers targeted 10 genes in the F0 generation and confirmed targeting by noting site-specific indels as a result of errors in NHEJ. They reported insertions as high +17, deletions as long as -23 (but the average was <10 on either extreme) and an efficiency of targeting up to 70% (but the average targeting rate was right around 30%).

Read more: Curing the world with CRISPR: Where we are at and where we ’re headed

The Rise of CRISPR babies

There have been many publications since improving the efficiency of Cas9 (Clements et al., 2017; Wolfs et al., 2016) as well as changing the functionality of the Cas9 enzyme itself (Esvelt et al., 2013; Gaudelli et al., 2017; Moreno-Mateos et al., 2017; Oakes et al., 2019; Shen et al., 2014), but the same issue remains with the CRISPR system itself, it can target a specific gene, but cannot precisely engineer a gene to have any sequence that we want. Because of this reality, this technology is not ready for use in humans. However, biophysicist Jiankui He completely breezed through all precautionary tape in November 2018 when he announced he had successfully engineered human babies using CRISPR at the 2018 Gene Editing Summit in Hong Kong.

Read more: The CRISPR gene-edited babies: a technological breakthrough or a brave new future?

He and his group planned to knockout the gene CCR5 in human embryos to help couples with HIV infected fathers conceive normal, healthy babies. He recruited these couples from an HIV/AIDS Support Group. Studies show mutations in CCR5 could make people more susceptible to influenza. CCR5 is also known to work in the immune system. He believes this will not affect cognitive ability, but studies in mice show the opposite (Zhou et al., 2016). 8 couples originally enrolled in this study (1 dropped out). All had father HIV+ and mother HIV-. Only 1 couple became pregnant, but the study had appeared to stop before the rest of the couples could do the same. However, during the question and answer session after his talk, He did mention there was another woman in the early stages of pregnancy.

The Science behind the CRISPR Babies

He and his colleagues designed their sgRNAs very carefully to minimize off-targets and only identified a single potential off-target site. They did not identify any off target sites in 19 embryos that were working with and the only mutations were found in CCR5. 31 embryos were injected with Cas9 protein and sgRNA, 70% were viable post injection. Preimplantation genetic diagnosis (PGD) confirmed CCR5 editing in 2/4 viable blastocysts. Thus, they started a two-embryo pregnancy. A 15-nucleotide deletion was confirmed. This is significant because it is not necessarily a true knockout because it is not a frame shift mutation. Nonetheless, the parents elected to move forward.

Notably, the babies are definitely mosaic. He already mentioned that CRISPR targeting did not begin until the 3-cell stage. By modifying these children at this stage of development, each cell does not necessarily have the same genotype. While mutations should be in the same region of CCR5, the mutations created by CRISPR are not precise and could vary extensively. He plans to follow up with the twins Lulu and Nana for the next 18 years to monitor their progress and the results of the CRISPR modifications.

Full Transcript of Jiankui He’s Talk

Ethical Concerns

Before these experiments even began, the study itself had some glaring issues. The need to create HIV resistant babies was unnecessary because there are already established methods such as HAART therapy to essentially eliminate the possibility of transmitting HIV from Mom to baby. The father being HIV+ really plays into it because the embryo doesn’t get HIV from creation. HIV comes from exposure to mothers’ blood for 9-months, though there is really good data that appropriate HAART therapy for both mom+baby makes transmission essentially zero. Honestly, I think its definitely patient abuse because he straight up said that he found the participants at an HIV support group. The couples likely didn’t know or were misinformed about the medical practices to protect a baby from an HIV infection. Thus, they thought this was the only way to have a healthy child.

He kept much of his work a secret. All couples were given informed consent and this process was not clearly reviewed by the proper channels. None of Jiankui He’s companies were involved in the funding of this study. He also paid all of the medical expenses for the couples during the process. Lastly, he hints at the couples receiving some small money on the side.

Moving Forward

Jiankui He’s work on creating genetically modified human twin girls is groundbreaking only in the sense that his group was the first to successfully genetically engineer human embryos with CRISPR. The work itself is not groundbreaking. Similar results have been reported in publications using CRISPR to modify model organisms such as mice and zebrafish. He and his colleagues are essentially treating human embryos like laboratory rats to perform experiments that show this technology can work in humans (which is not surprising) and offers no necessary medical advantage. This work was likely done to become the first to do such experiments and claim the notoriety. However, because of this, He has lost his job at the Southern University of Science and Technology in Shenzhen, his Ph.D. advisor Michael Deem and Stanford University (He’s PostDoc institution) is under investigation, and neither He nor Deem will likely be able to get another grant to do scientific research in their lifetimes. Nonetheless, this could be just the beginning. Many groups have already been performing on human cells and there could be others with plans to do similar work in human embryos.

Read more: Opinion: The first CRISPRed babies are here, what’s next?

Many individuals have tried do-it-yourself CRISPR outside the realms of academia and none of produced any significant results. Famously Josiah Zayner tried to increase his own muscle mass by using a DIY CRISPR kit (that he also sells online) to no such results. Bryan Bishop, famous for being an early investor in Bitcoin, is at the forefront of one of these rogue groups. A programmer by trade, Bishop looks at the human genome like a code that can be optimized. He believes that by using gene-editing technology, we can create humans that are not only resistant to genetic disease, but also engineered to be smarter, more moral, less prone to obesity, and be able to put on muscle mass much easier. Despite grossly underestimating the role of epigenetics in human development, Bishop’s approach as a BioHacker might be the most realistic.

What Bishop has that most of these groups do not is the capital. Bishop outsources all of his laboratory work to experts in the field (notably in Ukraine) working in academia. This has alarmed many experts in the field, especially since Bishop claims he already has a couple on board willing to try using CRISPR on their own offspring. Despite all of this, the experiments that are being done are focused on rats at the moment. Unlike Jiankui He, Bishop does not want to genetically modify one-cell stage embryos, but instead has focused on modifying sperm cells before fertilization. Recently, Bishop has shifted his plans into harvesting stem cells from one embryo (donor), modifying them, and then inserting these modified cells into another embryo (receiver). This very much resembles germ-cell transplants commonly done in model organisms like zebrafish and mice. While a feasible method, this has huge ethical concerns because one embryo must be sacrificed to genetically modify another.

Nonetheless, to resolve all of this, we shouldn’t institute an unnecessary ban on the technology, but instead continue to work on these tools to improve their efficiency and precision as well as work to have a sober conversation about the potential implications of this technology. Right now CRISPR has huge potential to drastically shape the future of humanity. We can very easily target genes and knockout their function. However, we still cannot precisely engineer the genome outside of point mutations. By continuing to work on improving this method, we should be able to precisely engineer any locus we desire and thus, in the future, the idea of a genetically modified person won’t sound so scary.

Thomas Clements, Ph.D. is a Lecturer in the Biological Sciences Department at Vanderbilt University. He graduated with his Ph.D. in Biochemistry and Cell Biology from Rice University in May 2018. His research centers on improving CRISPR-Cas9 gene editing in zebrafish and is continuing this work in a Discovery-Based Laboratory Course at Vanderbilt. He is also a member of the Early Career Scientist Policy and the Education Committee at the Genetics Society of America. He is passionate about science literacy and inspiring the next generation of scientists to pursue careers that align with their passions.

 

*Portions of this document can be found Thomas Clements’ Ph.D. Thesis at Rice University: “A Rice CRISPy Treat: Improving CRISPR-Cas9 gene editing in the zebrafish to facilitate analysis of genes implicated in neural angiogenesis in an F0 screen.” May 2018

References

Carrington, B., Varshney, G.K., Burgess, S.M., and Sood, R. (2015). CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity. Nucleic Acids Res. gkv802.

Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J.-W., and Xi, J.J. (2013). Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos. Cell Res. 23, 465–472.

Clements, T.P., Tandon, B., Lintel, H.A., McCarty, J.H., and Wagner, D.S. (2017). RICE CRISPR: Rapidly increased cut ends by an exonuclease Cas9 fusion in zebrafish. Genes. N. Y. N 2000 55.

Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819–823.

Esvelt, K.M., Mali, P., Braff, J.L., Moosburner, M., Yaung, S.J., and Church, G.M. (2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods advance online publication.

Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U. S. A. 109, E2579–E2586.

Gaudelli, N.M., Komor, A.C., Rees, H.A., Packer, M.S., Badran, A.H., Bryson, D.I., and Liu, D.R. (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature advance online publication.

Gonzales, A.P.W., and Yeh, J.-R.J. (2014). Cas9-based genome editing in zebrafish. Methods Enzymol. 546, 377–413.

Huertas, P., Cortés-Ledesma, F., Sartori, A.A., Aguilera, A., and Jackson, S.P. (2008). CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature 455, 689–692.

Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.-R.J., and Joung, J.K. (2013). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229.

Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., and Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429–5433.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821.

Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T.F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. Off. Publ. Am. Assoc. Anat. 203, 253–310.

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., and Church, G.M. (2013). RNA-Guided Human Genome Engineering via Cas9. Science 339, 823–826.

Moreno-Mateos, M.A., Fernandez, J.P., Rouet, R., Vejnar, C.E., Lane, M.A., Mis, E., Khokha, M.K., Doudna, J.A., and Giraldez, A.J. (2017). CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat. Commun. 8, 2024.

Oakes, B.L., Fellmann, C., Rishi, H., Taylor, K.L., Ren, S.M., Nadler, D.C., Yokoo, R., Arkin, A.P., Doudna, J.A., and Savage, D.F. (2019). CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification. Cell 176, 254–267.e16.

Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., Wang, L., Hodgkins, A., Iyer, V., Huang, X., et al. (2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods advance online publication.

Wei, C., Liu, J., Yu, Z., Zhang, B., Gao, G., and Jiao, R. (2013). TALEN or Cas9 – Rapid, Efficient and Specific Choices for Genome Modifications. J. Genet. Genomics 40, 281–289.

Wolfs, J.M., Hamilton, T.A., Lant, J.T., Laforet, M., Zhang, J., Salemi, L.M., Gloor, G.B., Schild-Poulter, C., and Edgell, D.R. (2016). Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease. Proc. Natl. Acad. Sci. 201616343.

Zhou, M., Greenhill, S., Huang, S., Silva, T.K., Sano, Y., Wu, S., Cai, Y., Nagaoka, Y., Sehgal, M., Cai, D.J., et al. (2016). CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory. eLife 5.

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