The Promise of Base Editing: Potential Applications in Science and Medicine

The Promise of Base Editing: Potential Applications in Science and Medicine
Posted in: Therapeutics

Introduction

The discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has revolutionized the field of molecular biology and medicine (reviewed by Kantor et al.). CRISPR-mediated genome editing initially involved the generation of a Cas9-induced double-strand DNA (dsDNA) break (Figure 1) repaired by either non-homologous end joining (NHEJ) mechanisms, or by homology-directed repair (HDR). Successful HDR would allow for specific alteration of the genome as desired, making this repair method the preferred one for therapeutic purposes. HDR can theoretically be harnessed to 

FIGURE 1. Depiction of CRISPR-Cas9-mediated double-strand cutting of a target DNA sequence. PAM, protospacer adjacent motif; sgRNA, single-strand guide RNA. Taken from Singh et al. Vet. Sci. 2021, 8, 122. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

insert a specific DNA template for precise restoration of the DNA sequence, but this pathway is characterized by limited efficiency and high rates of undesired insertion or deletion (indel) mutations that nullify the potential benefit resulting from repairing the mutation. Moreover, due to reliance on homologous recombination, HDR-mediated editing is restricted to dividing cell types, therefore limiting the range of diseases that can be targeted. Consequently, these issues have been addressed by the development of base editing without dsDNA cutting, as outlined below.

Base Editor Basics

There are two classes of DNA base editors: cytosine base editors (CBEs) and adenine base editors (ABEs) (Figure 2). All four transition mutations (C→T, T→C, A→G, and G→A) can be installed using these two classes of base editors. Anzalone et al.). Notably, over 25% of human pathogenic single-nucleotide polymorphisms (SNPs) can be corrected by targeting the four transition mutations.

A future blog in the Zone will discuss prime-editors (PEs), the latest additions to the CRISPR genome-engineering toolkit, expanding the scope of editing to include transversion mutations, as well as small insertion and deletion mutations. 

FIGURE 2. CRISPR DNA base-editing tools. DNA base-editors encompass two key components: a Cas9 “nickase” (nCas9) [or inactive “dead” Cas9 (dCas9)>

enzyme for programmable DNA binding, and a single-stranded DNA modifying enzyme for targeted nucleotide alteration. Two classes of DNA base-editors have been described: cytosine base-editors and adenine base-editors. Cytosine deamination generates uracil, which base pairs as thymidine in DNA. Fusion of uracil DNA glycosylase inhibitor (UGI) inhibits the activity of uracil N-glycosylate (UNG), thus increasing the editing efficiency of cytosine base-editing in human cells. Adenosine deamination generates inosine, which has the same base-pairing preferences as guanosine in DNA. Collectively, cytosine and adenine base-editing can install all four transition mutations (C→T, T→C, A→G, and G→A). Taken from Kantor et al. Int. J. Mol. Sci. 2020, 21, 6240. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Cytosine Base Editors (CBEs):

In 2016, Liu and co-workers developed the first-generation base-editor (CBE1) by fusing a cytosine deaminase to the catalytically deficient, or “dead,” Cas9 (dCas9) (Kantor et al., 2020). In a narrow window of the non-targeted strand, CBE1 deaminates C to uracil (U), which is then recognized by cell replication machinery as a T, resulting in a C-G to T-A transition. Importantly, although CBE1 mediates efficient, targeted base editing in vitro (up to 37% editing with a 1.1% indel formation rate), it is not effective in human cells. This inefficiency is mainly due to repair of the U-G intermediate by the base excision repair (BER) pathway. BER of U-G in DNA is initiated by uracil N-glycosylate (UNG), which recognizes the U-G mismatch and cleaves the glycosidic bond between U and the deoxyribose backbone of DNA, resulting in reversion of the U-G intermediate created by the base editor back to the C-G base pair. 

To improve base editing efficiency, a second-generation cytosine base editor (CBE2) was developed by fusing a uracil DNA glycosylase inhibitor (UGI) to BE1 to inhibit the activity of UDG. The inhibition of BER by BE2 resulted in a 3-fold increase in editing efficiency in human cells. 

To further improve editing efficacy, BE3 was developed by restoring histidine at position 840 in dCas9 to generate a base editor that uses Cas9 nickase (nCas9). This variant induces a nick in the G-containing strand of the U-G intermediate (non-edited DNA strand) to bias cellular repair of the intermediate towards a U-A outcome, further converted to T-A during DNA replication. This modification increased editing efficiency by 6-fold for BE3 over BE2. The use of nCas9 also exhibited an increase in indel frequency of 1.1% compared to 0.1% in BE2; however, this rate is still much lower than that induced by DSBs.

Further optimization of CBE was performed to reduce indel formation during base-editing, improve editing efficiency, and narrow the editing window. An improved fourth-generation cytosine base editor (CBE4) was generated by fusing an additional copy of UGI to the N terminus of nCas9 with an optimized 27-bp linker. Subsequent variations of these approaches to additional CBEs can be read about in the above-cited review by Kantor et al. 

Adenine Base Editors (ABEs): 

Epigenetic methylation of C-G base pairs in genomic DNA leads to 5-methylcytosine (5mC)-G base pairs, primarily in C-G rich promoter regions upstream of genes. Because 5mC is vulnerable to high rates of spontaneous C deamination to T leading to a transition mutation, this accounts for nearly half of all pathogenic point mutations. In principle, these relatively abundant mutations can be reversed using an ABE to convert a T-A mutation back to the non-mutant C-G base pair (Figure 2). As such, base editing capabilities and studies of genetic diseases were further expanded by the Liu Lab in 2017, with the development of ABEs. 

ABE-mediated DNA editing operates under a similar mechanism as CBE. The ABE-dCas9 fusion binds to a target DNA sequence in a guide RNA-programmed manner, and the deoxyadenosine deaminase domain catalyzes the conversion of A to inosine (I). During DNA replication, an I is read as a G, leading to replacement of the original A-T base pair with a G-C base pair at the target site (Figure 2). Unlike cytosine deaminases, ssDNA adenosine deaminase enzymes do not occur in nature. This limitation was cleverly overcome through extensive protein engineering and directed evolution of Escherichia coli tRNA adenosine deaminase, TadA. 

The first-generation ABE (ABE1.2) was generated by fusing the evolved TadA variant (TadA*) to nCas9 through a 16 amino acid linker used in BE3. In comparison with CBE, ABE yields a much more homogeneous DNA product that has virtually no indels and no significant off-target (A-to-non-G) edits. Nevertheless, optimizations continue to be made, furthering improvements to editing efficiencies and reducing off-target effects. 

For further discussion of CBEs and ABEs, check out the TriLink-sponsored webinar presented by CRISPR-Cas9 leader Professor David Liu. After discussing the molecular-level development of the various types of CBEs and ABEs, presenters discuss investigations that apply ABEs to correct progeria. 

Five Ways Base Editing Can Alter Genes 

There are five ways base editing is currently used to change genomes:

FIGURE 4. Five ways base editing is used to change genomes. Drawn by Jerry Zon based on public information provided by Beam Therapeutics.

Here are examples of each of these five strategies:

  1. Gene Correction: An A to T mutation in codon 6 of the HBB gene for the β chain of hemoglobin can cause sickle-cell disease. Editing T → A corrects this defect. (Newby et al.). 
  2. Gene Modification: Donor DNA comprised of wild-type codon 6 is inserted by CRISPR-Cas9 to correct a mutation in HBB that causes sickle-cell disease. (Dever et al.).
  3. Gene Activation: Re-activation of fetal γ-globin (HBG) gene expression, which is typically silenced after birth, can ameliorate the clinical course of β-hemoglobinopathies. (Ravi et al.).
  4. Gene Silencing: Early stop codons can be introduced in ∼17,000 human genes by the CRISPR-STOP method for gene knockout for research tools (Kuscu et al.).
  5. Multiplex Editing: Increasing CRISPR-Cas9 multiplex editing capability with the endogenous transfer RNA (tRNA)-processing system to include multiple sgRNAs (Xie et al.).

The Impact of Base Editing: 

Due to its incredible potential for rewriting genomes, it is no surprise that base editing has quickly attracted tremendous attention. The initial 2016 publication in Nature reporting CBE has currently received 137,000 accesses, while the 2017 report in Nature on ABE has currently received 161,000 accesses, collectively indicating a truly remarkable level of interest. In addition, the chart in Figure 3 shows the frequency of base editing publications in PubMed, demonstrating a nearly linear increase from four in 2016 to over 200 in 2020.

FIGURE 3. Frequency of base editing publications since 2016. PubMed search query ("base editing") OR ("base editor") and chart by Jerry Zon (September 6, 2021).

Clearly, the ability to use base editing in therapeutic strategies is of major interest to the scientific and medical communities. However, the scope of base editing applications can be expanded even more. Examples from animal breeding, plant breeding, and bacterial bio-production are detailed below. Additional areas of application include viruses, insects, fish, and fowl. While space limitations prevent detailed discussion of these topics, there are plenty of resources available, including many review articles, as linked above. 

Human Therapeutics

Synopses of genome base-editing therapies under development by three companies at the forefront of these efforts are described below.

Beam Therapeutics cofounders include base-editing guru David Liu and CRISPR guru Feng Zhang. Beam Therapeutics has a relatively diverse pipeline across delivery technologies, therapeutic areas, and stages of development. Their most advanced program, which is at IND enablement, involves electroporation to deliver BEAM-101 to treat sickle cell disease (SCD)/β-thalassemia by fetal hemoglobin activation. 

A single base pair alteration causes sickle cell disease (SCD) in the HBB gene for β-hemoglobin that leads to the inability of red blood cells to transport enough oxygen throughout the body. Described in Nature in 2021, Liu et al. corrected the HBB mutation to a non-pathogenic variant with extremely high efficiencies in mice. Their research showed a durable effect after 16 weeks, leading to a 5-fold decrease in red blood cell sickling. 

Editas Medicine cofounders include Beam cofounders Liu and Zhang and CRISPR-guru/Nobel Laurate Jennifer Doudna. Like Beam, Editas has a relatively diverse preclinical pipeline that includes SCD/β-thalassemia and CNS. Notably, there is also an early-stage clinical assessment of EDIT-101 to treat Leber congenital amaurosis type 10 (LCA10), a severe retinal dystrophy caused by mutations in the CEP290 gene.

EDIT-101 is a genome-editing therapeutic that removes an aberrant splice donor created by the IVS26 mutation in CEP290 in order to restore normal CEP290 expression. Sub-retinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing, with comparable success in a non-human primate model. Preliminary clinical trial results showed that 2 of 5 patients sensed more light than they could before treatment, and one of these patients had improved visual acuity 6 months after starting treatment. Three additional patients did not make clear gains. Side effects reported were mild. The LCA10 investigators have now begun to treat four more adults with the highest dose available in the trial. 

Verve Therapeutics was launched in May 2019 with the mission of developing base editing therapies to treat cardiovascular disease, specifically coronary artery disease, which is the most common form of heart disease and the leading cause of death worldwide. Verve has entered into a strategic collaboration with Beam Therapeutics to access Beam’s base editing, gene editing, and delivery technologies for certain cardiovascular targets. 

Currently, Verve’s pipeline lists single-course in vivo gene editing programs intended to turn off genes in the liver implicated in cardiovascular disease, and they strive to do so in a safe and durable manner. Programs targeting PCSK9 and ANGPTL3 are in the preclinical stages.

Livestock Applications

The potential benefits of CRISPR technology to livestock applications is vast and expanding: 

“This technology allows for modifications that lead to improvements in livestock production traits, animal health, and welfare, generation of more refined large animal models of human diseases, pharmaceutical protein production, and investigating gene function. Since 2014, over 500 research papers have been published using CRISPR gene editing approach[s>

in livestock (pigs, cattle, sheep, and goats; based on [an>

October 1st, 2020 PubMed search).” (Perisse et al., 2021)

Clearly, the possible uses in the livestock industry are diverse. Two of the arguably most impactful applications to humans include the expansion and improvement of food sources for the world’s increasingly large population, and organ xenotransplantation to alleviate shortages in human organs for transplant.

Sheep: In July 2020, Zhou et al. published the first-ever report on applying ABE in large animals, in which they generated sheep with targeted amino acid substitutions. In brief, the bone morphogenetic protein receptor 1B (BMPRIB) gene, first identified in Booroola merino sheep, was known to be a major factor associated with increased ovulation rates (Reader et al.). The FecBB mutation in BMPR1B is highly associated with increased ovulation rate and litter size in domestic sheep breeds. Zhou et al. used Liu’s improved ABE (termed ABEmax) to introduce the FecBB mutation in the genome of Tan sheep, a local Chinese breed. ABEmax mRNAs and sgRNA were co-injected into ovine one-cell stage zygotes, and developing embryos were then transferred into surrogate ewes. A much higher editing efficiency (75%) was obtained compared to the one obtained (23%) in a previously reported approach (Mahdavi et al.).

Pigs: According to a June 2020 review by Song et al., pigs (Sus scrofa) are important livestock animals that provide large quantities of meat worldwide. However, conventional trait selection and breeding still have two major vulnerabilities: slow genetic progress, and difficulty separating desired traits from undesired ones.Whole genome-wide association studies (GWAS) have been conducted with a large-scale SNP data set to dissect important genetic factors controlling traits of interest, such as meat quality and growth traits, reproduction, and virus resistance. Base editing can therefore be used to precisely target pig SNPs in order to obtain desired traits, as exemplified by the following reports. 

  • Zhang et al. used BEs to genetically delete three major glycan antigens, in an effort to reduce the immunoreactivity of porcine bioprosthetic heart valves extensively used for heart valve replacement in clinics. In this manner, it is possible to reduce the incidence of immune rejection of such valves in recipient patients.
  • Yuan et al. used CBEs to successfully introduce mutations in three SNP loci of interest in porcine blastocysts at an efficiency of 67%–71%, which is significantly higher than the editing efficiency of 4% using CRISPR-Cas9. These results suggest that CBEs provide a more straightforward and efficient method for improving economic traits, reducing breeding cycles, and increasing pig disease tolerance.
  • Xie et al. reported that multiplexed CBEs efficiently induced conversions of trios of genes simultaneously in two different cases. This not only demonstrated a strategy to accelerate the generation of animal models with multiple point-mutations, but also accelerated studies on gene therapies for genetic diseases. In one of these cases, the pig model lacked B cells, T cells, and NK cells, a promising notion for xenotransplantation.

Plant Breeding

A July 2021 review by Azameti and Dauda titled Base Editing in Plants: Applications, Challenges, and Future Prospects provides a comprehensive account with numerous references to the original publications cited and discussed therein. Another informative resource is the September 2020 review by Van Eck titled Applying Gene Editing to Tailor Precise Genetic Modifications in Plants.

Delivery: Before considering some examples of base editing in plants, it is important to recognize that one of the differences between plant and animal cells is that plants have a cell wall that is not penetrated by molecules or complexes greater than ~5 nm. Additionally, plant cells are resistant to transfection with plasmid DNA (pDNA) or mRNA complexes with carriers, such as LNPs, with diameters typically >30 nm. Nevertheless, according to Van Eck, plant genetic engineering has been achieved through various delivery methods, including floral dip, direct DNA uptake into protoplasts (i.e., after removal of the cell wall), particle bombardment (aka gene gun or biolistics), and Agrobacterium-mediated techniques (Figure 5). Each of these methods has advantages and disadvantages that need to be considered when choosing a delivery approach because not all methods work, or at least not efficiently enough for all plant species.

FIGURE 5. Examples of gene editing delivery methods. Taken from Monsur et al. Genes (Basel) 2020 Apr 24;11(4):466. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Representative Examples: The above-mentioned review byAzameti and Dauda provides a comprehensive (3 page) tabular list of 27 publications during 2017-2020 covering 10 different types of base editor constructs collectively targeting many loci in various plants, but mostly rice. Table 1 is an abbreviated list of these base-edited plants, the number of publications for each, and the phenotype/trait-improvement of interest:

Table 1. Summary of Various Base-Edited Plants as of June 2021 (Adapted from Azamati et al.)

PlantsNo. StudiesPhenotype/Trait
Rice26Herbicide resistance, high yield
Wheat2Herbicide resistance
Tomato1Herbicide resistance
Watermelon1Herbicide resistance
Cotton1Unspecified
Soybean1Unspecified
Oilseed rape1Herbicide resistance

As a final point, it should be mentioned that base editing of crops is offered as a service by companies that specialize in the genetic engineering of plants. Two examples of plant base-editing service providers are Lifeasible and Creative Biogene, both located in the US. 

Bacterial Bioproduction

Global climate change is challenging the future of human societies. To reduce pollution that drives climate change, we need a more sustainable food supply chain and greener fuel options. One possible solution is applying biotechnology to convert inorganic one-carbon (C1) gases, such as carbon dioxide (CO2) and carbon monoxide (CO), into protein, biofuels, and commodity chemicals (Xia et al.). Both gases are already available in huge quantities, in part due to greenhouse gas pollution and industrial waste gases (Figure 6). Many studies have found that the bacterium Clostridium ljungdahlii can convert CO2 and CO gases, in combination with hydrogen gas, into acetateand ethanol via the Wood-Ljungdahl pathway. The resulting acetate can be used for producing paints, coatings, and fabrics, while ethanol can be used as a gasoline additive and manufacturing solvent. 

FIGURE 6. Graphical depiction of gene-editing bacteria for C1 gases-to-commodities. Drawn by Jerry Zon.

To evaluate the feasibility of base editing C. ljungdahlii as a model for improving acetogenic bacteria, Xia et al. developed a modularized base-editing tool by coupling dCas9 from S. pyogenes with activation-induced cytidine deaminase from the sea lamprey Petromyzon marinus. To increase the editing efficiency, they fused UGI (Figure 2). Furthermore, a fusion to a Leu-Val-Ala protein degradation tag was added, resulting in an overall lower amount of the fusion protein in the cell, thus minimizing the potential toxicity of dCas9 and UGI. Finally, a tetracycline repressor-promoter (tetR-Ptet) system, inducible with anhydrotetracycline in C. ljungdahlii, was included for the regulated expression of the base-editing tool.

Using this tool, four genes in two different metabolic pathways involved in ethanol production in C. ljungdahlii were disrupted to reprogram the carbon flux for improved acetate production as a first application. Disruption involved editing to produce premature STOP codons in two pathways: from acetyl-CoA to ethanol, and from acetate to acetaldehyde. The results of various fermentation experiments wherein metabolites of interest were quantified and were consistent with the targeted reprogramming.  

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