Maliha Tanjum Chowdhury’s winning essay for Wellcome Sanger Competition 2019.

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“CRISPR-cas9: opportunities and hurdles for clinical translation”

The CRISPR-cas system is native across bacteria and archaea, and stands as a remarkable example of adaptive immunity in prokaryotes against viruses and extrachromosomal DNA. The system relies on incorporating part of the viral DNA into the host genome upon first encounter, and using the RNA transcribed from the viral DNA to target and degrade the virus via cas enzymes during subsequent infections. Since its discovery, scientists have adapted it to target and manipulate genes in various model systems as it can do so much more efficiently than previous gene-editing tools1. Gene knockout studies have never been this easy, but that is not nearly where it ends in terms of potential applications for CRISPR.

The CRISPR-cas system is native across bacteria and archaea, and stands as a remarkable example of adaptive immunity in prokaryotes against viruses and extrachromosomal DNA. The system relies on incorporating part of the viral DNA into the host genome upon first encounter, and using the RNA transcribed from the viral DNA to target and degrade the virus via cas enzymes during subsequent infections. Since its discovery, scientists have adapted it to target and manipulate genes in various model systems as it can do so much more efficiently than previous gene-editing tools1. Gene knockout studies have never been this easy, but that is not nearly where it ends in terms of potential applications for CRISPR.

CRISPR-cas9, the specific system found in and adapted from Streptococcus pyogenes among others, represents an extremely attractive avenue in the treatment of genetic disorders, typically those characterized by mutations in a single genetic locus. Studies conducted with animals, human embryos2, and in cell culture3 have shown great promise in the specificity and low risk of using CRISPR-cas9 (and several other cas enzymes) as an effective gene-editing tool. But promise is not enough when it comes to human application.

A recent study showed that in a mouse model of Duchenne muscular dystrophy, intravenous treatment with a CRISPR-containing vector targeting the defective dystrophin gene successfully restored protein function4. But the study also highlighted a few concerns about the technology. The researchers found that the mice were able to develop immune responses against the vector, and there was evidence of off-target mutations caused by cas9. These issues are neither isolated, nor are they the only potential roadblocks to clinical translation. While CRISPR-based treatments rely on homologous recombination, repair of cas9-mediated by non-homologous end joining can result in large deletions5. Editing of embryos have been shown to result in mosaicism6, where only some of the cells successfully undergo gene-editing. While not insurmountable, these challenges must be addressed before CRISPR can be used in humans.

The immunity problem The development of adaptive immune responses against cas9 in the mice with muscular dystrophy is not surprising. Multiple studies have found evidence of both preexisting immunity against cas9 proteins in human blood samples, with one study finding that 96% of the sampled individuals had T cell-based immunity against cas97,8. This is explained by the fact that the cas9 enzymes commonly in use come from bacteria that humans frequently encounter, such as Streptococcus pyogenes. This is a major problem because preexisting T cell-based immunity would result in immune attack against cells containing the cas9, potentially compromising any treatment. To get around this problem, other variants of the cas enzyme from bacteria that are less frequently encountered by humans could be used, or the structures of common cas9 variants could be manipulated to avoid pre-existing immunity. These strategies may still just work the first time around, as exposure to these proteins could then result in an adaptive immune response. But a single exposure may be enough for many gene-editing therapies. Another potential avenue that could be explored in clinical trials is coadministration of CRISPR-containing vectors with immunosuppressive drugs.

Aiming for perfection in precision and efficiency

Efforts have been ongoing to both find more precise natural cas enzymes, and edit common cas9 varieties to increase accuracy. One study, for instance, shows the effectiveness of a cas9 enzyme harvested from a lesser used bacterial strain – N. meningitidis – in being more specifically targeted to the desired site-of-edit9. In addition, recently discovered cas9 inhibitors, called “anti CRISPR” or Acr proteins have been identified in N. meningitidis which can be used as off-switches of the genome-editing mechanism when and where needed. Other studies show similar efforts in engineering highly efficient cas enzymes that have been enhanced to not only increase gene-editing activity but to also recognize several different PAMs and bring about multiplex gene alterations10.

Among other applications, CRISPR-cas9 may have to be administered to human IVF zygotes/embryos in individuals. The looming concern in this context is mosaicism, which may not only reduce viability of the zygote where IVF-zygote implantation itself is a rare phenomenon, but may also introduce risks of the therapy failing completely11. Possible solutions include administering CRISPR immediately after fertilization and halting before rapid mitotic proliferation, which has been shown to be effective in monkey embryos, and, alternatively, administering CRISPR-cas9 to the germline stem-cells of the parents so that their gametes are genetically edited before fertilization. But these problems may be avoided in most cases by screening embryos for “healthy” individuals, without the need for CRISPR intervention.

The China ‘designer-baby’ fiasco: futile fury, or justifiable outrage?

The final – and perhaps most difficult to resolve – dilemma that must be addressed before novel remedies can be clinically applicable is whether the technology involved, and its outcomes, are ethical and universally acceptable. And it is impossible to discuss this in the context of the CRISPR-cas9 system without mentioning the great debacle last year regarding the Chinese ‘CRISPR-twins’ – claimed to be the first gene-edited babies by the scientist who experimented on them, Dr. He Jiankui12. He administered CRISPR-cas9 to alter the CCR5 gene in the human embryos before implanting them into the mother’s uterus, who gave birth to twin daughters later. He claimed that mutating the gene in this manner would make the twins resistant to HIV. There was backlash against this from the scientific community for a number of reasons that highlighted the ethical as well as technical challenges of introducing CRISPR-based treatments in humans. The technical concerns included the evidence that one of the twins was a mosaic, which would mean that the HIV-resistance would not even be expressed if none of the edited cells go on to form the white blood cell lineage. In addition, the mutation that he introduced is not the same as the one that confers resistance against some strains of HIV in European populations, and is in fact, completely uncharacterized in literature. Dr. Jiankui essentially disabled a perfectly normal gene, the absence of which is known to increase susceptibility to West Nile virus and Japanese encephalitis, in a completely unpredictable manner.

The consent that was signed by the parents did not state outright the possible risks normally associated with CRISPR-cas9 gene-editing, much less that their children’s genes would be altered; instead it stated that this was a trial testing a novel vaccine against AIDS, and that the project team will not be responsible if there is alteration of other genes caused by this. The ethical violations are infuriatingly obvious; scientists underscore how sensitive an issue gene-editing is, as these altered genes will be passed down through generations and persist in the population. Should they have negative effects, they will therefore have given rise to novel hereditary disorders – a truly ominous irony. The misadventures of this rogue scientist12 could single-handedly extinguish the warmth of hope kindled by the prospect of CRSPR-cas9 technology in preventing genetic diseases due to very justifiable public horror, not to mention what would happen if other scientists are influenced by him. Responsible regulation will be indispensable in making such treatments available and acceptable to the public. Especially after such a frightful ruse regarding such promising technology, it is up to scientists, regulatory board(s) and the government to promote the benefits of CRISPR-cas9 as well as the robust strategies being employed in resolving the risks and uncertainties associated with it. They must make the public aware of the laws/regulations that are effective, as well as his/her rights and how they can be exercised when being the recipients of such delicate mode(s) of healthcare.

Tightening the lawful reigns

Several regulatory measures are in action to prevent any misuse of this newly emerging technology. In North America, Congress has prohibited the Food and Drug Administrations from ever soliciting clinical trials on human embryos, whereas the National Institutes of Health lawfully do not fund such research12. And, the National Academies of Science, Engineering and Medicine have announced conclusively in 2017 that gene-editing in human embryos should only and only be done when there are no possible/viable alternatives for a cure or improved quality of life. After the CRISPR-edited Chinese babies, there was a brief outburst of public anxiety regarding whether, in the future, CRISPR would be used to design people with intense intelligence, beauty or physical prowess. Such fears are, for now, exaggerated and baseless, since these qualities depend on the workings of several genetic and environmental factors in tandem. Regardless, there is no guarantee that it will not become a possibility with the advent of more sophisticated technology, when the application of CRISPR-cas stretches beyond the clinic. One thing to note is that the regulations that exist are based primarily in developed countries and take into account the ethics and necessities of only those nations. First and foremost, if the aspiration is to launch CRISPR as a generic gene-editing tool in the clinic, it should first be ensured that the people who will be receiving the treatment understand what is being done to their genes and that of their babies, along with the legitimate probability of this system failing and the real risks involved. It is high time that the public get educated about at least the most basic concept of CRISPR-based genetic modification. Moreover, the clinical technicians, doctors and other personnel who will be using this technology in all parts of the world must undergo rigorous and internationally standardized academic and practical training to become eligible enough to provide service. Social scientists must work hand in hand with scientists in the regulatory bodies to gauge public opinion (e.g. by doing surveys and, say, open-ended campaigns involving syphoning public viewpoints and FAQs) to ensure the development of more well-rounded, generally popular policies. Thus, if a more internationally spanning set of laws and regulations – which consider the ethical, economic and health requirements of lesser developed countries – are constituted and accepted unanimously, then the transitioning of CRISPR-cas to the clinical platform will be smoother and everlasting.

Opportunities galore

Say what we may, but contemporary research still has piles of evidence to show that CRISPR-cas9, in the long run, may not be so dark a force as some fear, but a beacon of hope in giving people whose lives would have otherwise been shadowed by disability, exclusion and substantial social and financial burden, a perfectly healthy life. Several studies on the use of CRISPR in human germ cells and embryos have been conducted and show satisfactory results in removing faulty alleles in heterozygous mutants. One notable study demonstrated that CRISPR could fix a defective MYBPC3 allele which causes cardiomyopathies in individuals2. Another study employed CRISPR-cas9 to disable a gene in human blood progenitor cells which normally discourages hemoglobin production in fetuses, thus inducing increased hemoglobin production in this rapid-growth stage of life. This could counter the harmful effects of β-hemoglobinopathies13.

Even with all the challenges described above, the promises of this novel technology outweigh the risks; the seemingly strong push for CRISPR-cas9 to finally enter the clinical stream is highlighted by the recent collaborative venture of two companies which, namely, Vertex Pharmaceuticals, based in Boston and CRISPR Pharmaceuticals, a Swiss company which have labs based in Cambridge, Massachusetts13. The companies are running clinical trials of CRISPR-cas9 in Europe, and will soon do the same in the US. It appears that other companies are soon to follow suit. It has barely been six years since the technology was first developed. These are important milestones in the clinical application of gene therapy, and while it is untrue that the fundamental risks associated with CRISPR-cas9 technology can be simply side-stepped, the challenges have been identified and are in the process of being addressed.

[Words: 1,943]

Maliha Tanjum Chowdhury,

Maliha Tanjum Chowdhury,

Undergraduate student at the School of Life Sciences (SLS),

Independent University, Bangladesh (IUB),

Dhaka, Bangladesh.

  1. 1. J Ayub Med Coll Abbottabad. 2019 Jan-Mar;31(1):108-122. Advances In Research On Genome Editing Crispr-Cas9 Technology. Shah SZ1, Rehman A1, Nasir H1, Asif A2, Tufail B3, Usama M4, Jabbar B5.
  2. Nature. 2017 Aug 24;548(7668):413-419. doi: 10.1038/nature23305. Epub 2017 Aug 2. Correction of a pathogenic gene mutation in human embryos. Ma H1, Marti-Gutierrez N1, Park SW2, Wu J3, Lee Y1, Suzuki K3, Koski A1, Ji D1, Hayama T1, Ahmed R1, Darby H1, Van Dyken C1, Li Y1, Kang E1, Park AR2, Kim D4, Kim ST2, Gong J5,6,7,8, Gu Y5,6,7, Xu X5,6,7, Battaglia D1,9, Krieg SA9, Lee DM9, Wu DH9, Wolf DP1, Heitner SB10, Belmonte JCI3, Amato P1,9, Kim JS2,4, Kaul S10, Mitalipov S1,10.
  3.  Mol Genet Genomics. 2017 Jun;292(3):525-533. doi: 10.1007/s00438-017-1299-z. Epub 2017 Mar 1. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Tang L1,2, Zeng Y3, Du H3, Gong M4, Peng J4, Zhang B4, Lei M3, Zhao F5, Wang W6, Li X7, Liu J8.
  4.  Nat Med. 2019 Mar;25(3):427-432. doi: 10.1038/s41591-019-0344-3. Epub 2019 Feb 18. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nelson CE1,2, Wu Y1, Gemberling MP1,2, Oliver ML1, Waller MA1,2, Bohning JD1,2, Robinson-Hamm JN1,2, Bulaklak K1,2, Castellanos Rivera RM3, Collier JH1, Asokan A4,5, Gersbach CA6,7,8.
  5.  Published: 16 July 2018. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Michael Kosicki, Kärt Tomberg & Allan Bradley Nature Biotechnology volume 36, pages 765–771 (2018).
  6.  Published: 15 January 2019, Pages 156-162, Volume 445, Issue 2, ScienceDirect Mosaicism in CRISPR/Cas9-mediated genome editing. Maryam Mehravara, Abolfazl Shiraziab, Mahboobeh Nazaric, Mehdi Banand.
  7.  Published: 29 October 2018. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population, Dimitrios L. Wagner, Leila Amini, Desiree J. Wendering, Lisa-Marie Burkhardt, Levent Akyüz, Petra Reinke, Hans-Dieter Volk & Michael Schmueck-Henneresse. Nature Medicine volume 25, pages242–248 (2019).
  8.  Published: 30 October, 2017, Wiley Online Library, Immunity to CRISPR Cas9 and Cas12a therapeutics, Wei Leong Chew.
  9.  Genome Biol. 2018 Dec 5;19(1):214. doi: 10.1186/s13059-018-1591-1. NmeCas9 is an intrinsically high-fidelity genome-editing platform. Amrani N1, Gao XD1, Liu P2,3, Edraki A1, Mir A1, Ibraheim R1, Gupta A2,4, Sasaki KE1,5, Wu T2, Donohoue PD6, Settle AH6,7, Lied AM6, McGovern K6,8, Fuller CK6, Cameron P6, Fazzio TG9,2, Zhu LJ9,2,10, Wolfe SA2,3, Sontheimer EJ11,12.
  10.  Nat Biotechnol. 2019 Mar;37(3):276-282. doi: 10.1038/s41587-018-0011-0. Epub 2019 Feb 11. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Kleinstiver BP1,2,3,4,5, Sousa AA1,2,3, Walton RT1,2,3,5, Tak YE1,2,3,4, Hsu JY1,2,3,6, Clement K1,2,4,7, Welch MM1,2,3, Horng JE1,2,3, Malagon-Lopez J1,2,3,4,8,9, Scarfò I2,10,11, Maus MV2,10,11, Pinello L1,2,4,7, Aryee MJ1,2,4,7,8, Joung JK12,13,14,15.
  11.  Published: 15 March, 2017, NewScientist, Mosaic problem stands in the way of gene editing embryos: The first results of gene editing in viable human embryos reveals it works better than we thought, but that there’s another big problem blocking the way. Michael Le Page.
  12.  Published: December 5, 2018. Why Are Scientists So Upset About the First Crispr Babies? Only because a rogue researcher defied myriad scientific and ethical norms and guidelines. We break it down. By Gina Kolata and Pam Belluck. The New York Times.
  13.  US Companies Launch CRISPR Clinical Trial: The Germany-based study will test an ex vivo genome-editing therapy for the inherited blood disorder β-thalassemia. Published :Sep 3, 2018, by Catherine Oxford, TheScientist.