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CRISPR Base Editors: An Upgrade for Treatment of Genetic Disease

The CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9(CRISPR-associated system 9) technology, often touted as one of the greatest recent inventions to drive progress in biotechnology, is currently being challenged and viewed more critically as a recent flurry of articles is casting doubt on the overall safety of this rising technology which up to now has mostly seen positive press.

CRISPR gene editing recent news and controversies

CRISPR, also often referred to as CRISPR/Cas9, is a technically and scientifically fascinating biological system that promises to yield a revolution in treatment of genetic diseases. CRISPR also offers a basis for powerful and flexible tools for furthering our understanding of eukaryotic gene expression. The underlying biology – though rather obscure just a decade ago – is nowadays an enormously active area of research in both basic science and medical/technical applications.


CRISPR has such a promise that a cautionary article about issues with off-target genetic mutations in some CRISPR applications (Kosicki et al., 2018), also recently presented in the Wall Street Journal (WSJ) by Mohan and Marcus (2018) shook the research community. Both the study in question and the industry effects that the WSJ article touch upon are relevant when using the form of technology called CRISPR/Cas9 (Cas9 DNA nuclease paired with its targeting CRISPR RNA(s)). However, there are more advanced and specialized versions of CRISPR technology that may avoid those challenges, in this case referring to the ‘base editor’ versions of derived Cas9 (Komor et al., 2016, Nishida et al., 2016). More about the details of both will be discussed later in this post.

Image credit:  Cancer Research UK.

Recent “ruling by the European Court of Justice that gene editing equals genetic engineering”

The concerns over use of CRISPR-Cas9 in the context of therapeutic development have been further amplified with a recent ruling by the European Court of Justice that classified gene editing as genetic engineering. The new ruling now will also apply to treating genetic disease in humans and to genetically altering animals, and consequently all “edited organisms” will need to be labeled as GMOs (Genetically Modified Organisms).

In spite of the emerging challenges related to potential safety issues, the science keeps moving forward: A wide range of gene-editing applications are currently researched, all of which rely on various CRISPR-Cas9 techniques including

  • specific DNA cutting,
  • CRISPR modifications that avoid DNA cutting,
  • a modified CRISPR system that switches one DNA letter for another without cutting the DNA,
  • or the use of inactivated Cas9 fused to other enzymes that turn genes on or off.

CRISPR is a prokaryotic immune system

Some of the features of the CRISPR system were discovered decades ago and have recently been elegantly reviewed by Adli (2018). Initially, the CRISPR system was noted by the presence of its repeated element and spacer sequences in E. coli. It was later discovered that CRISPR sequences are broadly distributed being present in more than 40% of bacteria and 90% of archaea. Another key observation was the finding that spacer sequences were derived from phages/viruses and mobile genetic elements. This hinted at the fact that CRISPR could be an adaptive immunity system for prokaryotes. In an evolving scheme based on the diversity of active components and mechanisms there are currently two mechanistic classes divided into at least six types of CRISPR systems in prokaryotic cells. Much development work is now focused on the relatively simpler type II, within class 2, especially from Streptococcus pyogenes, which is characterized as having one active nuclease, Cas9 (Adli, 2018 and Wang et al., 2016).

CRISPR a powerful tool for gene editing

In the landmark paper by Jinek et al.(2012), it was shown that the S. pyogenes Cas9 protein in complex with two small RNAs called CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) will bind to specific DNA sequences and create a double strand break in that target DNA. The specificity of the cutting comes from the complementarity of part of the crRNA sequence binding to the target DNA as well as the presence of the PAM (protospacer-adjacent motif) sequence just 5’ to the target sequence in the complementary strand. In this way, prokaryotic organisms have a way to disrupt and eliminate foreign DNA elements via double-stranded breaks. The same paper also found that the activity of Cas9 could be fully supported by a single chimeric RNA, later called a single-guide RNA (sgRNA), which contains parts of both crRNA and tracrRNA.

CRISPR’s emergence as a powerful tool for genetic engineering evolved soon after in a series of papers where it was shown that the S. pyogenes Cas9 activity could be reconstituted in eukaryotic cells and specifically retargeted by altering the sequence of the sgRNA(s) (Jinek et al.(2013), Mali et al.(2013), and Cong et al.(2013))This was done via the expression of an optimized version of the Cas9 protein along with sgRNA(s) in human cells.

Double-stranded breaks in DNA, an opportunity and a problem

Since these initial descriptions, there has been an explosion of modified and derived systems that use Cas9. These applications are too numerous and varied to describe here (for excellent recent reviews see Adli (2018) and Wang et al. (2016). Strictly speaking when considering genomic DNA editing there are potential problems as described in the opening paragraphs. This is due to the fact that when double-stranded breaks are made in the DNA the lesions are corrected by multiple endogenous systems in eukaryotes, including non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is prone to indel formation, as well as creation of inversions and translocations of the DNA. HDR has typically fewer of these issues especially when paired with a repair template, but operates at lower efficiency. When multiple off-target sites can be bound by sgRNAs these repair activities can induce undesired mutations which can be problematic in the context of potential applications such as gene therapy for disease. Even when the sgRNA leads to targeting the correct site, there still may be many unwanted larger scale insertions, deletions, or rearrangements (Kosicki et al., 2018).
Although there is still significant disagreement about how much these unwanted mutations occur in varying context this represents a significant issue that will need to be closely evaluated going forward.

Base editor CRISPR

In the drive to be able to efficiently correct point mutations researchers developed derived versions of Cas9 where the protein was fused to one of several cytidine deaminases. These forms of Cas9 which have the ability to convert cytidine to uracil in the target DNA, and upon replication and repair cause C-to-T transversions are known as ‘base editors’ (Komor et al., 2016, Nishida et al., 2016). These Cas9 versions are based on either catalytically inactive Cas9 (termed dCas9) where both DNA cutting domains have been inactivated or nCas9 for nickase Cas9 where one DNA cutting domain is inactive. For both forms, the modifications are a way to navigate around off-target problems with the double-strand break inducing Cas9. It was particularly ingenious to design the nCas9 form that can nick the strand opposite to the one targeted by the deaminase so that the deaminated strand is preferentially selected as the donor for repair. In addition, the more advanced cytidine base editors incorporate a Uracil Glycosylase inhibitor (UGI) module to further reduce host elimination of the created U residue before repair can take place. More recently, an adenine base editor (ABE) form of Cas9 has been created (Gaudelli et al., 2017) where an evolved tRNA adenine deaminase has been fused to Cas9. The ABE can deaminate adenine in the DNA to inosine which when repaired is read as a G. Therefore, with these base editors it is now possible to specifically direct both point mutation transitions (purine for purine and pyrimidine for pyrimidine) in very precise ways in target DNA in eukaryotes.

Companies pursuing CRISPR technology in research and disease

crisprtheraAmong companies involved in CRISPR research and mentioned in the recent WSJ article is CRISPR Therapeutics founded by Emmanuelle Charpentier, one of the original developers of the CRISPR technology. They are headquartered in Zug, Switzerland with R&D operations in Cambridge, Massachusetts. CRISPR Therapeutics is working on ex vivo treatments for hemoglobinopathies β-thalassemia and sickle cell disease. Their product CTX001 will involve taking a patient’s own hematopoietic stem cells and using CRISPR/Cas9 to increase expression of the fetal hemoglobin gene. CTX110, on the other hand, is a product that uses CRISPR/Cas9 to help create allogeneic anti-CD19 CAR-T cell therapies to treat cancers. A review of CRISPR Therapeutics’ current pipeline also shows that they plan to use CRISPR gene editing to directly correct various genetic defects in vivo, with potential applications in cystic fibrosis, hemophilia, and glycogen storage disease Ia.

editasAnother company actively pursuing the CRISPR technology for therapeutics is Editas Medicine which was founded in 2013 and is headquartered in Cambridge, MA. The company’s research focuses on blood, muscle, liver, lung and eye diseases. One early product candidate termed EDIT-001 uses an AAV5 vector to deliver two sgRNAs and Staphylococcus aureus Cas9 to correct the CEP290 intronic mutation c.2991+1655A>G by inversion or deletion of intervening DNA, with the goal to enable treatment of Leber Congenital Amaurosis Type 10.

intelliaIntellia Therapeutics was founded in 2014 and also has its headquarters in Cambridge, MA. Jennifer Doudna is a co-founder and was one of the original scientists to describe the CRISPR/Cas9 system’s functionality. Intellia is pursuing both ex vivo and in vivo applications for CRISPR in treatment of disease. One relatively more advanced effort involves attempting to treat transthyretin amyloidosis (ATTR) by disrupting the transthyretin (TTR) gene which is often mutated in the disease. The approach towards treatment of ATTR in humans is via the use of lipid nanoparticle formulation to deliver the CRISPR agent in order to disrupt the TTR gene in the liver. Overproduction of mutant protein by the liver accounts for a significant amount of disease pathology.

caribouCaribou Biosciences is another company, co-founded by Jennifer Doudna and Martin Jinek, that aims to bring CRISPR technology to bear on multiple fronts. Their aims include use of CRISPR in therapeutic context as well as agricultural, industrial, and basic biology research. Caribou actually had a hand in the founding of Intellia to further its goals in medicine. Based on recent publications and examination of the company website the current focus seems to be broadly on methods development in the field of CRISPR.


Adli, M.The CRISPR tool kit for genome editing and beyond (2018) Nat Commun, 15(9):1911.

 Gaudellli et al. Programmable base editing of A•T to G•C in genomic DNA without DNA Cleavage. (2017) Nature, 551(7681):464-471.

 Jinek et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity (2012) Science, 337(6096):816-821.

Jinek et al. RNA-programmed genome editing in human cells. (2013) eLife, 32:e00471.

 Komor et al. Programmable editing of a target base in genomic DNA without double-stranded cleavage. (2016) Nature, 533(7603):420-424.

Kosicki et al. Repair of double-stranded breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. (2018) Nat Biotechnol, 36(8):765-771.

Cong et al. Multiplex genome engineering using CRISPR/Cas systems. (2013) Science, 339(6121):823-826.

Mali et al. RNA-guided human genome engineering via Cas9. (2013) Science, 339(6121):819-823.

 Mohan and Marcus. New research prompts selloff in companies using CRISPR technology (2018) WSJ, July 16, 2018

 Nishida et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. (2016) Science, 353(6305):aaf8729.

Wang et al. CRISPR/Cas9 in Genome Editing and Beyond. (2016) Annu Rev Biochem, 85:227-264.


Nick Marshall

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