A Bespoke Genome: The Future of Human Gene Editing

A Bespoke Genome: The Future of Human Gene Editing

Tags: , ,

By: Aravin Sukumar

In March 2017, researchers at Temple University, Philadelphia, demonstrated the ability of the CRISPR-Cas9 gene editing technology to eliminate latent human immunodeficiency virus-1 (HIV) provirus in mice.1 HIV infection targets immune cells, in particular CD4+ T-cells, which leads to a progressive failure of the immune system and ultimately to acquired immunodeficiency syndrome (AIDS).2 When T-cells are infected, HIV RNA is converted into DNA and integrated into the host genome, referred to as provirus, which is essential for replication. In some cells, the HIV provirus remains inactive and escapes anti-retroviral therapy. The provirus can become activated unexpectedly thereby preventing the complete elimination of the virus. Although this study was conducted in mice to demonstrate the elimination of the HIV provirus through gene editing, there is hope that its immense therapeutic potential will eventually be translated to humans.

Human gene editing toolbox

Three types of gene editing tools are currently employed that can delete or insert anywhere between single nucleotides to large kilobase sequences: 1) zinc-finger nucleases (ZFN); 2) transcription activator-like effector nucleases (TALEN); and 3) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9.3 In principle, these tools all consist of an engineered DNA-targeted nuclease that acts as “molecular scissors” to create double-stranded breaks (DSB) in the genome, which then triggers one of two distinct DNA repair mechanisms: homologous recombination (HR) or non-homologous end joining (NHEJ). When a homologous DNA template is present, HR-mediated repair can insert new sequences or introduce single-nucleotide polymorphisms (SNPs). In contrast, when a DNA template is absent, NHEJ produces random small insertions, deletions, or inversions surrounding the DSB, which can contribute to gene inactivation. The inspiration for employing gene editing in HIV treatment stemmed from a patient who was homozygous for a 32-base pair deletion in the gene CCR5, the major co-receptor for HIV entry, and whose T-cells were therefore resistant to infection.4 To mimic this patient’s protective deletion, Tebas et al. (2014) designed ZFNs to disrupt the function of CCR5 in T-cells derived from HIV patients and evaluated their resilience to infection. Interestingly, infusion of CCR5-modified T-cells in patients resulted in a lower decline in T-cells, thereby demonstrating the promising potential of gene editing as a treatment for HIV. In fact, both ZFNs and TALENs have already been tested as potential treatment target in HIV, while the application of the CRISPR-Cas9 has only just begun.

CRISPR-Cas9: a bacterial defense system

The most recent gene editing tool is the CRISPR-Cas9 system, developed through discoveries originating from work in bacteria.5 This system was initially identified in bacteria as a form of adaptive immunity against foreign DNA from plasmids or viruses (bacteriophages), but has been harnessed in mammalian cells to perform permanent genome modifications. CRISPR genomic loci are distributed extensively throughout the bacterial genome. They are comprised of a set of CRISPR-associated (Cas) genes, which encode RNA metabolism enzymes and the molecular scissors—Cas9 protein—followed by a series of palindromic repeat sequences (“direct repeats”) interspaced by variable sequences (“spacers”) corresponding to foreign genetic elements. These CRISPR loci are transcribed as a precursor CRISPR RNA (pre-crRNA) and processed by the encoded metabolic enzymes into mature, short CRISPR RNAs (crRNA) that can interact with and direct the Cas9 enzyme to cleave foreign nucleic acids containing sequences found in the “spacers.”

Harnessing CRISPR-Cas9 in human cells

Several researchers are credited with advancing the CRISPR-Cas9 system in mammalian cells. In 2012, Drs. Jennifer Doudna and Emmanuelle Charpentier engineered a single guide RNA (sgRNA) capable of recruiting the Cas9 protein to cleave DNA at a specific site.6 They were also able to program the site-specificity of Cas9 by modifying the seed sequence of the sgRNA—a sequence complementary to the target sequence and essential for binding to the genome. Building upon these important discoveries, Dr. Feng Zheng from the Broad Institute has confirmed the application of the CRISPR-Cas9 system in mammalian cells by transfecting the CRISPR machinery into human and mouse cell lines and evaluating the efficacy and sites of DNA cleavage.7 The importance of these landmark findings was recognized by the scientific community through numerous awards, including Canada’s most prestigious international 2016 Gairdner Award, presented to Drs. Doudna, Charpentier, and Zheng, as well as to other pioneers in the field, including Drs. Rodolphe Barrangou and Phillipe Horvath.8

Future of CRISPR-Cas9: promises and pitfalls

When compared to previous gene editing strategies such as ZFN and TALENs, the CRISPR-Cas9 system has more far-reaching applications and displays unparalleled versatility. For example, the system facilitates simpler and more effective development of transgenic animal and cell-based models to simulate disease processes, thereby benefiting researchers in medical sciences.9 Furthermore, the agricultural industry has also adopted CRISPR-Cas9 techniques to improve traits of livestock and enhance crops’ resistance to pests. Lastly, the system has a tremendous therapeutic potential in monogenic disorders such as Huntington’s disease and cystic fibrosis, whose incurable prognosis may, in the future, be altered by gene editing therapies.

Notwithstanding the enormous potential of CRISPR-Cas9 to medicine, many safety, ethical, and socio-economic issues are raised with respect to its applications in humans, echoing concerns regarding DNA recombination in the mid-1970s. One of the major safety concerns with CRISPR-Cas9 is the possibility for non-specific DNA modifications that could potentially inactivate essential genes or activate oncogenic genes.10 CRISPR-Cas9 also raises ethical questions pertaining to its use in the genetic modification of human embryos or germ cells (sperm or oocyte), a process that is criminally banned in Canada and other parts of the world.11 In 2015, an international dialogue to discuss the scientific, ethical and governance issues of human genome editing took place in Washington, DC.12 This meeting, termed the International Summit on Human Genome Editing, was co-hosted by the National Academy of Sciences and Medicine (US), the Chinese Academy of Sciences (China), and the Royal Society (UK). A report highlighting the outcomes of this meeting was released in February, outlining recommendations for the future use of gene editing in humans.10 It concluded that basic research on gene editing is necessary to fully understand the scope of the current technology and to maintain adequate oversight on clinical trials in human somatic cells. More importantly, it was recommended that gene editing in human germ cells not be performed for enhancement of traits, and should only be allowed for compelling reasons until we gain a better understanding of whether the benefits outweigh the long-term risks.

Considering its immense potential impact on mankind—from preventing and treating disease to enhancing traits like cognitive or athletic ability—gene editing has been described as a “process more rational and quicker than Darwinian evolution.”12 Evidence offered by Yin et al. (2017) and many others supports the notion that CRISPR-Cas9 genome editing in humans is plausible and offers great hope for millions affected by previously untreatable diseases.1 Yet, since the potential risks are not readily apparent, it would be wise to proceed with caution in this new era of genomic medicine. Moving forward, we must collaborate globally on gene editing research and, while abiding by the legal and regulatory policies, allow researchers the freedom to explore the potential of CRISPR-Cas9 and its potential to prevent and cure disease.


  1. Yin C, Zhang T, Qu X, et al. In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Mol Ther. 2017;25(5):1168-1186.
  2. Kaminski R, Chen Y, Fischer T, et al. Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing. Sci Rep. 2016;6:28213.
  3. Drake MJ and Bates P. Application of gene editing technologies to HIV-1. Curr Opin HIV AIDS. 2015;10(2):123-127.
  4. Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370(10):901-910
  5. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-1278.
  6. Jinek M, Chylinksi K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.
  7. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819-823.
  8. Semeniuk, I. Nature’s Scissors. The Globe and Mail [newspaper online]. 2016 Mar 24 [cited 2016 May 28]. Available from: https://www.theglobeandmail.com/technology/science/gairdner-awards-honour-gene-editing-crispr-researchers/article29330742/
  9. Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nat Biotechnol. 2016;34(9):933-941.
  10. Human Genome Editing Science, Ethics, and Governance – Report Highlights: National Academy of Sciences and Medicine. 2017. Available from: http://nationalacademies.org/cs/groups/genesite/documents/webpage/gene_177260.pdf.
  11. Knoppers BM, Isasi R, Caulfield T, et al. Human gene editing: revisiting Canadian policy. NPJ regenerative medicine. 2017;2:3.
  12. International Summit on Human Gene Editing – Meeting Summary: National Academy of Sciences and Medicine. 2015. Available from: https://www.nap.edu/catalog/21913/international-summit-on-human-gene-editing-a-global-discussion.