CRISPR/Cas-mediated DNA base-editing in gene therapy targeting β-hemoglobinopathies

By Milena Flankova


CRISPR-Cas-mediated DNA base-editing is a step up from the famous CRISPR/Cas9 genome editing platform. Base-editing is capable of modifying haematopoietic stem and progenitor cells (HSPCs). This approach is paramount when silencing the genetic regulators such as BCL11A which affects the expression of β– and γ-globin genes in β-hemoglobinopathies – sickle cell disease and β-thalassemia. High performance rates of the base-editors instill hope in everyday use of these techniques in the clinical setting.


The original gene therapy technique employed for hemoglobinopathies was based on the use of allogeneic haematopoietic stem cell (HSC) transplantation from human leukocyte antigen (HLA) matched donors (Locatelli et al., 2013). However, efficiency of this approach is restricted due to a limited availability of immunocompatible donors. Allogeneic stem cell transplants bear the  risk of a recipient developing graft-versus-host disease (GVHD) which denotes exogenous HSCs attacking surrounding cells (Styczyński et al.., 2019). Research was consequently refocused ongene modification of autologous haematopoietic stem and progenitor cells (HSPCs), meaning HSPCs collected from the same individual for gene editing. 

CRISPR/Cas9 is one of the most promising gene editing techniques for modifying autologous HSPCs as it was shown to have a relatively high gene-targeting efficiency and adaptability (Mali, Esvelt and Church, 2013). Furthermore small guide RNA sequences (sgRNA) can be manipulated in order for cas9 to recognise different target sequences, compared to transcription activator-like effector nuclease (TALEN) and zinc-finger nuclease (ZFN) where an enzyme has to be reengineered. Additionally,CRISPR/Cas9 allows for multiple loci to be modified simultaneously (Cong et al., 2013). Naturally found Cas9-like effector proteins are typically large in size and hence are difficult to introduce into a vector. Discovered smaller Cas9 variants need more sophisticated protospacer adjacent motif (PAM) sequences implying lower targeting specificity (Kantor, McClements and MacLaren, 2020). Moreover, double-strand DNA breaks created by Cas9 nuclease generally contribute to off-target cutting and produce insertions or deletions (indels) that result in shortened, nonfunctional proteins which does not justify gene editing aiming to correct a mutation (Chakrabarti et al., 2019). 

DNA base-editing was established following the CRISPR/Cas approach but does not require double-strand break activity for introducing changes in the DNA sequence which signifies higher accuracy. This method relies on nitrogenous base replacement by a modified Cas9 protein which is fused with another enzyme which performs deamination of either adenine or cytosine (Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering, 2020) .

Following allogeneic hematopoietic stem cell transplantation (HSCT) approach, it can be difficult to find immunocompatible donors with sufficient HLA genotype matching. Moreover, allogeneic HSCT is associated with increased levels of infections, toxicity, GVHD (Styczyński et al., 2019)  and rejection. Briefly, autologous stem cell-based gene therapy involves the following steps. HPSCs are obtained from a patient either from iliac crests or through leukapheresis, a process of isolating white blood cells, including HSCs, from the rest of the blood. Gene editing is performed ex vivo and prior to reintroduction of a vector in vivo, usually using adeno-associated virus (AAV) vectors with CRISPR-mediated base-editing approach, patient’s endogenous HSPCs are destroyed by chemotherapy. This gene therapy technique is a promising treatment for β-hemoglobinopathies which are part of hemoglobinopathies as these are disorders caused by a single point mutation which can be easily manipulated using CRISPR-based editing methods (De Luca et al., 2019). 

β-hemoglobinopathies – the molecular overview

β-hemoglobinopathies are the most common monogenic disorders in the world, affecting more than 300,000 births annually. These are inherited disorders and include β-thalassemia, seen in roughly 20 % of patients with β-hemoglobinopathies, and more commonly seen, in around 80 % of the cases, sickle cell disease (SCD) (Modell, 2008). Both diseases are caused by mutations in hemoglobin beta (HBB) gene. In the case of β-thalassemia, the synthesis of a β-globin chain which links to a heme group in hemoglobin is limited. Additionally, it was determined that β-thalassemia can be caused by more than 400 mutations (Yang et al., 2020).  The majority of these mutations include single nucleotide deletions, substitutions or insertions. In SCD, hemoglobin is polymerised and red blood cells possessa C-shaped morphology which impairs the ability of red blood cells to carry oxygen and often results in clogging of blood vessels (Ikawa et al., 2019).Pathophysiology of SCD most commonly implicates a A-to-T point mutation in the HBB gene, whereby valine is substituted by glutamic acid. 

Since the last century it was known that patients with β-hemoglobinopathies develop symptoms that are less severe when levels of fetal hemoglobin (HbF) in red blood cells are increased. This was discovered by examining patients who also had a condition called hereditary persistence of fetal hemoglobin (Perrine et al., 1972). Moreover, inactivity of the BCL11A gene was found to be linked to elevated HbF levels. Hence down-regulating the BCL11A gene which normally suppresses HbF production can help mitigate the severity of these diseases. The transcription factor BCL11A is also a negative regulator of the γ-globin gene. Thus, BCL11A gene and its promoter tend to be targeted by various gene editing methods (Lamsfus-Calle et al., 2020). 

The principle behind CRISPR/Cas

CRISPRs or clustered regularly interspaced short palindromic repeats were first identified in 1987 in E.coli (Ishino et al., 1987). CRISPRs are repetitive DNA sequences present in genomes of prokaryotes like archaea and bacteria that are separated by spacers. Spacer DNA sequences in CRISPR loci were detected in approximately 40 % of sequenced bacterial genomes and in 90 % of genomes of archaea (Kunin, Sorek and Hugenholtz, 2007). These spacer sequences are derived from bacteriophage genomes, providing adaptive immunity to prokaryotes, whereby Cas endonucleases cleave DNA near the target sequence identical to spacers (Barrangou et al., 2007; Hille et al., 2018). Cas genes were detected uniquely in genomes containing CRISPRs. Moreover, CRISPR loci and cas genes were found to be closely located in the DNA sequence, denoting a functional importance (Jansen et al., 2002). Spacers of sequence repeats in each CRISPR locus are the result of Cas protein separating phage DNA into smaller sequences, referred to as protospacer sequences, and inserting them into its own genome. The CRISPR system is composed of two elements – a Cas protein and a guide RNA (gRNA). gRNA contains a CRISPR sequence with spacers, called CRISPR RNA (crRNA), and a trans-activating RNA (tracrRNA) which makes up a “scaffold”, linking crRNA to Cas. When the CRISPR-associated Cas protein recognises a protospacer adjacent motif (PAM) sequence in the target genome which is located downstream of the protospacer, it attempts to align it with gRNA. The Cas protein unwinds the DNA and gRNA hybridises with a protospacer. Once a match is found for a particular part of the DNA sequence, the Cas endonuclease cleaves both strands of the target DNA sequence, creating a double-strand break (Wiedenheft, Sternberg and Doudna, 2012; Cong et al., 2013; Kantor, McClements and MacLaren, 2020). The formation of double-strand breaks produces indels at a relatively high frequency which significantly reduces the benefit from correction of the initial point-mutation (Pinzon-Arteaga et al., 2020), as seen in hemoglobinopathies.

The outline of CRISPR/Cas-mediated base-editing

Base-editing is a novel approach to correcting point mutations which does not rely on double-strand breaks. The method involves fusing CRISPR/Cas9 with a deoxynucleoside deaminase enzyme which remains applicable with a gRNA, resulting in a direct conversion of one nitrogenous base to another. The nuclease activity of Cas9 can be inactivated by Asp10Ala and His840Ala mutations. The resulting catalytically “dead” Cas9 (dCas9) is still capable of associating with gRNA but cannot cleave the DNA backbone anymore (Komor et al., 2016). Typically used base-editors include cytosine base-editors (CBE) and adenine base-editors (ABE) which mediate conversion of C-to-T (Zhang et al., 2020)  and A-to-G (Gaudelli et al., 2017) respectively. That way, mutations involving all four nitrogenous bases in the DNA can be achieved. 

Cytosine base-editors

Initially, the first-generation cytosine base-editor’s efficiency was relatively low due to the base excision repair (BER). Cytidine deaminase converts cytosine to uracil, which is detected by cell replication machinery as thymine, producing a C-G to T-A mutation. The maximum base-editing efficiency obtained was 37 % due to a reversed base correction caused by BER, resulting in no overall change in the DNA sequence (Komor et al., 2016). Uracil N-glycosylase (UNG), a BER enzyme, recognises a U-G mismatch and replaces inserted U with C  during DNA replication (Kunz, Saito and Schär, 2009). Second- and third-generation cytosine base-editors were engineered to fuse with an uracil DNA glycosylase inhibitor (UGI) to the C-terminus of the first-generation cytosine base-editor model which contained dCas9 or Cas9 nickase (nCas9), thus suppressing the activity of uracil DNA glycosylase. Three-fold higher efficiencies were obtained with the second-generation base-editor in comparison to the first-generation base-editing system. The third-generation base-editor involves re-introducing His840 back to the dCas9, and hence enabling nicks of the non-edited DNA strand at guanine complementary to cytosine which was replaced by uracil. Nevertheless, this base-editor was still carrying the Asp10Ala mutation in the modified Cas9 endonuclease, which inhibits double-strand DNA break formation, and retained UGI fused to UNG. The experiments demonstrated that the third-generation base-editor has a two- to six-fold higher base-editing efficiency compared to the second-generation base-editor, exhibiting a permanent correction within the range of 15 to 75 % of total cellular DNA. Moreover, all of the developed base-editors showed less than 0.1 % of indel formation rates which is substantially lower than was caused by the original CRISPR/Cas gene editing. These results were comparable both in U2O and HEK293T human-derived cells (Komor et al., 2016).

Adenine base-editors

Adenine base-editor is used to install an A-T to G-C mutation. This base-editor works similarly to the cytosine base-editor. Adenosine deaminase catalyses the conversion of adenine to inosine which is recognised by DNA polymerases as guanine. Initially, there were studies done on RNA, as no single-stranded DNA adenosine deaminases were found to exist in nature. Experiments of editing on DNA with RNA adenosine deaminases were not successful. The fusion of the N-terminus of nCas9 to the modified E. coli-derived tRNA adenosine deaminase (TadA) responsible for converting inosine to adenine resulted in the first-generation adenine base-editor. The fusion was performed through 16-amino acid residue XTEN linker which was previously utilised in base-editing systems for priming potential deamination sites. Future generation adenine base-editors were developed through engineering TadA enzyme and thus altering its activity towards the DNA editing site. It was determined that adenine base-editors show reduced compatibility with Cas homologs (Gaudelli et al., 2017; Losey, Ruthenburg and Verdine, 2006) . Though some homologs like SaCas9, SpCas9-NG and CpCas9s are compatible with adenine base-editors, the resulting editing efficiencies were lower compared to those of cytosine bae-editors. This led to the development of PACE selection system with the aim to ameliorate the activity and compatibility of different Cas homologs with respect to adenine base-editing. Phage-assisted continuous evolution (PACE) method was able to solve these issues and additionally, it expanded the targeting scope (Richter et al., 2020). Latest adenine base-editors demonstrated up to 50 % of editing efficiency in human cells, exhibiting a 99.9 % product purity with negligible indel formation rates and off-target edits already from early generations (Gaudelli et al., 2017) .

Figure 1. The molecular mechanism of installing mutations in the DNA by using cytosine and adenine base-editors (Kantor, McClements and MacLaren, 2020).

Dual base-editing

Recent research was focused on generating an efficient dual CRISPR base-editing system operating with both cytidine and adenosine deaminases as this would make gene-editing more productive. The dual technique was found to have an increased activity on cytosines and a lowered activity on adenines. Thus, there is still a lot to improve (Zhang et al., 2020). 

Base-editing for correcting β-hemoglobinopathies

B-hemoglobinopathies could potentially be someday cured by CRISPR/Cas-mediated base-editing technique. Single base-editing techniques are capable of introducing only four transition mutations: C-to-T, G-to-A, A-to-G and T-to-C. Up to this day, these technologies are not able to perform the eight transversion mutations, such as C-to-A, C-to-G, G-to-C, G-to-T, A-to-C, A-to-T, T-to-A, and T-to-G. for instance, the T•A-to-A•T mutation in the HBB gene is required to correct the most typical cause of the SCD (Anzalone et al., 2019). However, adenine base-editors replace the opposite stranded adenine residue of valine with guanine. Consequently, valine is converted to alanine on that position, leading to the formation of a naturally occurring variant hemoglobin (Hb) G-Makassar. This mutation prevents polymer formation and SCD patients with Hb G-Makassar exhibit normal red blood cell morphology and hematological parameters. Moreover, these results are achieved with up to 70 % productive modification of the target adenine in autologous fibroblasts (Lin et al., 2019). In β-thalassemia patients, 114C-to-T or 113A-to-G mutations in the promoters of HBG1 and HBG2, which are γ-globin genes, suppressed expression of these genes by interfering with BCL11A binding site. This mutation promoted higher HbF levels and limited symptoms of the disease (Wienert et al., 2018).


Recent base-editing technologies have great advantages over the original CRISPR/Cas genome engineering method, leading to substantially higher editing efficiencies. Although base-editing has already come a long way and has shown to act upon β-hemoglobinopathies, reducing the symptoms, there are no universal base-editors which are capable of installing all possible mutations, particularly transversion mutations. Furthermore, all steps of gene therapy, including gene editing, vector-based modifications of HSPCs, cell culture and transduction, are equally important to investigate in order to ameliorate the success of engraftment. Lastly, ex vivo autologous HSPC therapy is costly and this factor limits the scope of research and the use of the technique in the clinic.


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