CRISPR base editing technology is a promising genome editing tool as (i) it does not require a DNA template to introduce mutations and (ii) it avoids creating DNA double-strand breaks, which can lead to unintended chromosomal alterations or elicit an unwanted DNA damage response. Given many cancers originate from point mutations in cancer-driving genes, the application of base editing for either modelling tumour development, therapeutic editing, or functional screening is of great promise. In this review, we summarise current DNA base editing technologies and will discuss recent advancements and existing hurdles for its usage in cancer research.

The advent of RNA-guided programmable clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases has revolutionized genome editing applications [1–4]. The ability of CRISPR–Cas9 to introduce desired edits in the genome with ease and great precision has accelerated the pace of genetic research. However, introducing precise point mutations remains technically challenging. As a large number of diseases, including many cancers, are caused by single nucleotide variants (SNVs), the ability to either install disease-driving point mutations for disease modelling and drug discovery, or reverse those point mutations for therapeutic applications, is of critical importance. Hence, the development of CRISPR base editors represent a potential solution for the ability to install point mutations and to correct disease-changing mutations [5].

CRISPR base editors avoid DNA double-strand breaks generated by nucleases, and therefore undesired chromosomal alterations. In addition, base editing technology circumvents the need for homology-directed repair, which relies on the addition of a DNA template and is inefficient in non-dividing cells [6]. Owing to its potential to introduce point mutations at ease, base editing enjoys great popularity, which is reflected by the pace of development ever since the first base editors were described. Those advancements and optimisations are crucial to facilitate the therapeutic applications for many diseases, including cancer [7].

While this review discusses base editing in mouse and human cells, base editing has been successfully applied in plants [8,9] — including cotton [10], rice [11], soybean [12] — zebrafish [13,14], pigs [15,16], rats [17], rabbits [18–20], sheep [21,22], and more recently monkeys [23,24].

In this review, we will highlight some of the recent advancements that have been made towards the application of CRISPR base editing in mammalian cells using different delivery methods of base editing reagents for disease modelling, therapeutic applications and for functional genetic screens. While the principles can be translated to other research and disease backgrounds, this review focuses on DNA base editing applications for introducing cancer-specific gene variants.

Originally DNA base editors were designed by creating Cas9 fusion proteins with enzymatic domains of proteins known to induce deamination of bases in DNA [25]. Directed to its target site in the genome by a specifically designed guide RNA, base editors install point mutations without the need for a template or introducing DNA double-strand breaks. There are two main DNA base editors developed to date enabling the conversion of all four DNA base pairs: Cytosine base editors (CBE) and Adenine base editors (ABE) [5].

Cytosine base editors

Komor et al. [25] from the Liu laboratory developed the first class of CBEs containing a fusion protein of rat Apobec1 protein linked with a catalytically dead version of Cas9, capable of introducing point mutations (BE1). A major hurdle to overcome for cytosine base editing is the cellular base excision repair (BER) pathway as deamination of cytosine to uracil leads to the excision of uracil by uracil DNA glycosylase (UDG). Therefore a uracil glycosylase inhibitor (UGI) from the Bacillus subtilis bacteriophage PBS1 was fused to the first CBE to protect the UG intermediate [25,26]. This next-generation base editor was called BE2. Indeed, the importance of BER for editing outcomes has recently been shown in cells with high BER function, which exhibit low base editing efficiency [27]. In a subsequent optimisation step, a nickase version of Cas9 fused to Apobec 1 and two copies of the UGI was used to create the commonly used BE3 CBE. The nicking of the unedited strand initiates mismatch-repair (MMR) as G is now flagged as a mismatch to U (which is read as T). This ensures the use of the edited strand as template for the repair, thereby introducing a permanent C-to-T mutation [25].

Adenine base editors

Adenine deaminating enzymes do not exist in mammalian cells. However, Liu and colleagues [28] identified a prokaryotic enzyme called TadA, which is known for its deaminating capability. Hence, to develop ABEs bacterial TadA deaminase domains were fused to a nickase version of Cas9. Subsequent optimisation rounds found the presence of one WT and one evolved TadA domain fused to a nickase version of Cas9 known as the common ABE7.10 base editor to be most efficient for A-to-G editing [28]. As the deamination of adenine results in inosine, and given inosine is not readily excised and therefore reversed by BER or any other cellular repair mechanisms, additional components to aid adenine base editing are not needed. Indeed, adenine base editing is achieved with great efficiency accompanied with very low rates of InDel production when compared with CBEs [29].

Mode of action

The ability of both classes of base editors to introduce point mutations relies on protospacer-adjacent motif (PAM) and single-guide RNA (sgRNA) mediated binding of the DNA by Cas9, which leads to denaturing of the DNA creating the typical R-loop thereby exposing a short stretch of single-stranded DNA on the non-complementary strand. The single exposed DNA strand is now accessible for deamination and subsequent introduction of the desired point mutation. For the widely used BE3 editor the editing window is 5 nucleotides in length, usually from positions 4 to 8 within the protospacer sequence, when position 1 is counted as first position 5′ upstream of the PAM site. The editing window for the commonly ABE7.10 editor is slightly tighter, ranging from positions 4 to 7 within the protospacer sequence, when the PAM is counted as positions 21–23 (Figure 1).

Composition of DNA base editors.

Figure 1.
Composition of DNA base editors.

Schematic of the basic structure of CRISPR DNA base editors. Cytosine base editors (CBE) consist of a nickase Cas9 variant (Cas9*) fused with a cytidine deaminase enzyme. In addition, one or two copies of an uracil glycosylase inhibitor (UGI) are fused with the protein complex to prevent cellular base excision repair (BER) mechanisms. CBEs introduce a C-to-T point mutation into DNA with a C:U intermediate. For adenine base editors (ABE) Cas9* is fused with an deoxyadenosine deaminase enzyme enabling a permanent A-to-G point mutation in DNA with a A:I intermediate. The target base is shown in the single-stranded R-loop. Diagram indicating the targeting window for CBE or ABE, with deamination positions between position 4–8 (CBE) and 4–7 (ABE) on the protospacer sequence, when the PAM site is counting as positions 20–23.

Figure 1.
Composition of DNA base editors.

Schematic of the basic structure of CRISPR DNA base editors. Cytosine base editors (CBE) consist of a nickase Cas9 variant (Cas9*) fused with a cytidine deaminase enzyme. In addition, one or two copies of an uracil glycosylase inhibitor (UGI) are fused with the protein complex to prevent cellular base excision repair (BER) mechanisms. CBEs introduce a C-to-T point mutation into DNA with a C:U intermediate. For adenine base editors (ABE) Cas9* is fused with an deoxyadenosine deaminase enzyme enabling a permanent A-to-G point mutation in DNA with a A:I intermediate. The target base is shown in the single-stranded R-loop. Diagram indicating the targeting window for CBE or ABE, with deamination positions between position 4–8 (CBE) and 4–7 (ABE) on the protospacer sequence, when the PAM site is counting as positions 20–23.

It should be noted that since its first inception, the design and efficiency of base editors has been continuously improved based on the original design of CBEs and ABEs mentioned above. For example, narrowing or widening the targeting window and minimising off-target effects have been a major focus of base editor engineering. While the description of those variants is beyond the scope of this manuscript, we would like to direct the readers to a recently published overview by the Liu laboratory [30].

The first generation of base editors have been established in standard cell lines, such as human embryonic kidney (HEK) cells [25,28]. However, delivery of CRISPR base editing reagents into primary cells or whole organisms remains challenging and presents the bottleneck of many base editing applications. Here, we summarise current attempts to install cancer-driving mutations in disease-relevant cells (Figure 2).

Overview of the different base editor delivery options useful for diverse applications.

Figure 2.
Overview of the different base editor delivery options useful for diverse applications.

Schematic showing different delivery options for base editors. Delivery of base editors into cells can be achieved by either direct injection or transfection of base editor constructs in form of mRNA, ribonucleoprotein (RNP) or plasmid. In addition, viral delivery options can be employed for either hard-to-transfect cells or intra-tissue delivery of base editors.

Figure 2.
Overview of the different base editor delivery options useful for diverse applications.

Schematic showing different delivery options for base editors. Delivery of base editors into cells can be achieved by either direct injection or transfection of base editor constructs in form of mRNA, ribonucleoprotein (RNP) or plasmid. In addition, viral delivery options can be employed for either hard-to-transfect cells or intra-tissue delivery of base editors.

Nucleic acid and RNP approaches

Because of its ease (cost and stability), plasmid encoded base editors are a convenient tool to achieve the expression of base editors after transfection into cells. Indeed, plasmids have been used in initial studies to establish base editing in cells and are used to enable in vivo editing by hydrodynamic tail vein injection. However, plasmids are toxic for some cell types, and, prolonged expression from plasmids can lead to undesired edits [31,32]. Another suitable delivery method is complexing Cas9 protein with synthesised sgRNAs to generate ribonucleoprotein (RNP) complexes [33]. Indeed, base editor RNP complexes have been successfully used for therapeutic editing in primary cells, such as human CD34+ cells [34]. Instead of RNPs, base editor constructs can be delivered as mRNA. Using mRNA offers the advantage that it can be readily translated into the base editor protein, and while mRNA can be chemically stabilised, it usually is degraded quickly. This reduces potential off-target effects while maintaining precise on-target editing [32]. Indeed, transfection of base editor mRNA together with sgRNAs targeting one or multiple genes by electroporation or lipofection appears to be a promising approach to achieve precise editing in primary cells, such as T cells or human induced pluripotent stem cells (hiPSCs) [29,35,36].

In addition to transfection techniques, gene editing components can be directly delivered into cells by microinjection. Indeed, to date fertilised oocytes have been successfully used for the generation of disease models by injection of base editor mRNA or RNP alongside gene targeting sgRNAs [37,38].

Viral delivery

Viral vectors have been the gold standard delivery vehicle for in vivo genome editing. In this regard, Adeno-associated viruses (AAVs) are widely used for local tissue delivery of genome editing tools [39]. However, owing to the packaging limits of AAVs (∼4.7 kb), delivery of Cas9 base editors via AAVs remains challenging. Recently, split AAV approaches have been described in which the coding sequence for base editors is split up in two AAV vectors and hence viral particles [40–43]. By utilising split-intein protein moieties base editor expression is achieved after viral delivery and trans-splicing of the protein products encoded by the dual AAVs [40]. In addition to AAVs, lentiviral vectors are commonly used to deliver CRISPR–Cas9 complexes into cells to generate cancer-driving mutations in vivo [44]. In this regard, Zafra et al. [45] recently delivered optimised versions of the BE3 CBE into human cancer cell lines. Similarly, in a recent pre-print publication, lentiviral vectors were used to achieve CBE expression in cancer cells for large-scale functional screening [46].

While viral delivery techniques ultimately might reach packaging capacities, the recently discovered novel Cas Θ enzyme presents an exciting avenue, as this novel Cas protein is much smaller in size compared with Cas9 while maintaining its ability to bind DNA [47].

An overview of how base editors are used with different delivery methods can be found in Table 1.

Table 1.
Summary of base editors and delivery modes for clinically relevant cells
Delivery typeBase editorDelivery routeModelReference
AAV CjABE (Campylobacter) Intracranial Glioblastoma [49
AAV BE3, ABEmax Retro-orbital Neurodegeneration [74
AAV ABE7.10, extended sgRNAs Intramuscular Muscular Duchenne Dystrophy [75
AAV BE3 Intravenous in utero; retro-orbital; vitellein vein for fetuses Therapeutic editing of hereditary tyrosinemia type1 [76
AAV SaKKH-BE3 tail vein injection Metabolic liver disease [40
AAV AID-CBEmax Intracochlear Mouse model of deafness (Baringo mouse) [41
AAV BE3 Tail vein injection Therapeutic editing of cardiovascular disease mouse model [77
AAV BE3 Intratracheal Therapeutic editing of ALS mouse model [42
AAV, plasmid BE4max Tail vein injection Metabolic liver disease [78
lentivirus BE3 Cell transduction BRCA1/2 screening [61
lentivirus BE3.9max Cell transduction Gene variant screening [46
RNP ABE Electroporation CAR-T cell engineering [56
RNP A3A (N57Q)-BE3 Electroporation Therapeutic SCD editing [34
RNP BE3 Zygote electroporation Embryo targeting [37
mRNA BE3, ABE7.10 2-cell stage injection Embryo targeting [52
mRNA BE4, ABE7.10; BE2, BE3, SaBE3, VQR-BE3, BE4, ABE Zygote injection Embryo targeting [51,37,53
mRNA ABEmax Transfection hiPSCs, patient-derived fibroblasts [79
mRNA ABE8 Electroporation CAR-T cell [57
mRNA BE4, ABE7.10 Lipofection hiPSCs [29
mRNA RA6.3 Lipid nanoparticle (LNP) tail vein injection Tyrosinemia mouse model [73
mRNA, plasmid ABE6.3, RA6.3 Tail vein injection Hereditary tyrosinemia type 1 [80
mRNA, RNP BE3, BE4, coBE4 Electroporation CAR-T cell [36
Plasmid BE3, BE3-FLNS Tail vein injection Hepatocellular carcinoma [45
Plasmid ABEmax, xCas9-ABEmax Transfection Human intestinal organoids, CF [81
Plasmid ABEmax-NG Electroporation Patient-derived liver and intestinal organoids [72
Transgenic expression BE3 Intraductal lentiviral sgRNA Triple negative breast cancer [48
Delivery typeBase editorDelivery routeModelReference
AAV CjABE (Campylobacter) Intracranial Glioblastoma [49
AAV BE3, ABEmax Retro-orbital Neurodegeneration [74
AAV ABE7.10, extended sgRNAs Intramuscular Muscular Duchenne Dystrophy [75
AAV BE3 Intravenous in utero; retro-orbital; vitellein vein for fetuses Therapeutic editing of hereditary tyrosinemia type1 [76
AAV SaKKH-BE3 tail vein injection Metabolic liver disease [40
AAV AID-CBEmax Intracochlear Mouse model of deafness (Baringo mouse) [41
AAV BE3 Tail vein injection Therapeutic editing of cardiovascular disease mouse model [77
AAV BE3 Intratracheal Therapeutic editing of ALS mouse model [42
AAV, plasmid BE4max Tail vein injection Metabolic liver disease [78
lentivirus BE3 Cell transduction BRCA1/2 screening [61
lentivirus BE3.9max Cell transduction Gene variant screening [46
RNP ABE Electroporation CAR-T cell engineering [56
RNP A3A (N57Q)-BE3 Electroporation Therapeutic SCD editing [34
RNP BE3 Zygote electroporation Embryo targeting [37
mRNA BE3, ABE7.10 2-cell stage injection Embryo targeting [52
mRNA BE4, ABE7.10; BE2, BE3, SaBE3, VQR-BE3, BE4, ABE Zygote injection Embryo targeting [51,37,53
mRNA ABEmax Transfection hiPSCs, patient-derived fibroblasts [79
mRNA ABE8 Electroporation CAR-T cell [57
mRNA BE4, ABE7.10 Lipofection hiPSCs [29
mRNA RA6.3 Lipid nanoparticle (LNP) tail vein injection Tyrosinemia mouse model [73
mRNA, plasmid ABE6.3, RA6.3 Tail vein injection Hereditary tyrosinemia type 1 [80
mRNA, RNP BE3, BE4, coBE4 Electroporation CAR-T cell [36
Plasmid BE3, BE3-FLNS Tail vein injection Hepatocellular carcinoma [45
Plasmid ABEmax, xCas9-ABEmax Transfection Human intestinal organoids, CF [81
Plasmid ABEmax-NG Electroporation Patient-derived liver and intestinal organoids [72
Transgenic expression BE3 Intraductal lentiviral sgRNA Triple negative breast cancer [48

Table showing different base editors for usage in clinically relevant cells. AAV: Adeno-associated virus; RNP: ribonucleoprotein; ALS: amyotrophic lateral sclerosis; BRCA1/2: breast cancer genes 1 and 2; CAR: chimeric antigen receptor; SCD: sickle cell disease; hiPSCs: human induced pluripotent stem cells; CF: cystic fibrosis.

Base editing technologies represent a breakthrough for the characterisation of cancer-driving mutations in vivo. Recently, colleagues from the Jonkers laboratory established a transgenic Cre recombinase inducible BE3 base editor system to test critical tumour-driving mutations in a mouse model of triple-negative breast cancer [48]. Using their cancer model which is driven by loss of p53 and BRCA1 in mammary tissue, cancer-driving point mutations, such as in the AKT/PI3K pathway, can be readily introduced after lentiviral intraductal delivery of sgRNAs.

In a different study, Zafra et al. [45], explored the importance of point mutations in driving hepatocellular carcinoma. By tail vein injecting plasmids of CBEs, BE3 or an optimised version with an additional N-terminal flag-tagged nuclear localisation signal (FNLS-BE3), together with plasmids encoding the Myc oncogene and sgRNA targeting the activating S45F mutation of the catenin-beta 1 (CTNNB1) gene to drive cancer development by increased WNT-signalling, tumours were driven in livers of adult mice. Interestingly, analysis of tumour nodules revealed near-complete editing for the activating S45F mutation [45].

In another attempt to improve disease modelling and therefore potential modes of drug discovery, the Kuehn laboratory tested the possibility of introducing tumour-driving point mutations in hiPSCs to generate a pool of readily available isogenic cells from healthy donors with the same genetic background [29]. Of particular importance are mutations in the TP53 gene for which more than 5000 mutations are currently described. Indeed, Surun and colleagues successfully introduced TP53 point mutations by lipofection of mRNA for CBE and ABE base editors alongside sgRNAs targeting seven missense and two nonsense mutations of the TP53 gene into hiPSCs. Functional analysis of those introduced mutations revealed that iPSCs with mutated p53 protein are more resistant to cell cycle arrest when treated with the small-molecule murine double minute (MDM2) inhibitor Nutlin-3a, thereby demonstrating the oncogenic potential of p53 mutations [29]. While they achieved editing efficiencies of up to 90% (Y163H mutation), they found ABE editing more efficient and accurate when compared with CBE editing [29].

In addition to those ascribed studies pointing to the ability of base editing technologies to drive cancer progression, the feasibility of in vivo base editing to correct a tumour-driving mutation has been tested [49]. Li et al. targeted the activating hotspot −124C > T mutation within the TERT promoter region driving glioblastoma development. Intracranial AAV-delivery of a nickase version of Campylobacter jejuni (Cj) Cas9 fused to the commonly used ABE ABE7.10, creating CjABE together with a sgRNA targeting the −124C > T mutation, impaired tumour growth in PDX models in the brains of mice treated with sgRNA targeting the −124C > T mutation [49]. This was accompanied by a reduction in TERT activity, increased apoptosis and interestingly in vivo editing efficiencies matched the ones established in vitro [49]. In summary, this elegant study establishes the possibility for targeting cancer-driving mutations in patients.

Another critical tool for mimicking disease, are murine models generated by direct injection of CRISPR reagents into fertilised oocytes [50]. While traditional HDR methods using Cas9 nucleases are successfully applied for the generation of mouse models, base editing technology might be advantageous if introducing a point mutation in a critical exon. Indeed, several studies used base editors to generated novel disease models by injecting either CBE or ABE encoding mRNA together with sgRNAs into the one-cell stage mouse embryos to introduce specific point mutations [51,52]. Detailed analysis of injected mouse embryos revealed substantial off-targets for CBE but not for ABE editing, probably owing to spurious deamination of random single-stranded DNA by the Apobec molecules of the CBEs [53].

Base editing holds great promise for genetic engineering of CAR-T cells aiding cancer immunotherapy. In contrast to conventional HDR methods using WT Cas9 nucleases, base editing can efficiently disrupt gene expression without leading to Cas9-mediated DNA double-strand breaks and subsequent chromosomal rearrangements [54].

In a pilot study, Webber et al. [36] used a codon-optimised 4th generation cytosine base editor (coBE4) for efficient multiplex base editing in primary CD3+T cells for genes relevant for the generation of allogenic CAR-T cells. Current CAR-T cell approaches for cancer therapy are hampered by the immune escape of tumour cells by up-regulating the immune checkpoint protein programmed death ligand 1 (PD-L1) to counteract programmed cell death 1 (PD1) receptor expression on CAR-T cells, thereby stalling its cytotoxic tumour-killing potential [55]. In the recent study, delivering coBE4 mRNA by electroporation together with sgRNAs targeted to disrupt splice sites for the T-cell receptor alpha (TCAR), beta-2 microglobulin (B2M) and PD-1 genes, led to an efficient reduction in protein levels in cells engineered with the CAR T cell receptor by lentiviruses. Importantly, those multiplex-edited T cells retained their functionality enabling target cell killing and due to the lack of PD-1 surface expression were able to overcome PD-1/PD-L1 induced tumour cell resistance [36].

Another example of overcoming PD-1/PD-L1 mediated tumour resistance, was shown by using adenine base editing to enhance cytotoxic function of CAR-T cells. Base Editing was used to reduce the N-linked glycosylation of PD-1 thereby decreasing its expression levels [56]. Similar to a genetic disruption of PD-1 expression in CAR-T cells, decreasing protein stability by altering the post-translational glycosylation pattern of PD-1 offers a robust avenue to overcome the inhibitory effect of tumour cells on immune cells. To this end, lentiviral co-delivery of sgRNA targeting the codon of amino acid N47 of the PD-1 protein (PDCD1 gene) and the coding sequence of the mesothelin-directed CAR into T cells was followed by electroporation of ABE protein into these cells. Shi et al. [56] investigated the anti-tumour functions of edited CAR-T cells in vivo and found that tumour growth was reduced leading to improved survival, which was due to greater expansion of base edited CAR-T cells with decreased surface expression of PD-1.

Recently, Gaudelli et al. [57] generated a newer edition of ABE editors (ABE8s) and tested their potential to induce precise point mutations in primary T cells from healthy donors. Electroporating ABE8 mRNA into CD3+CD28+ activated T cells together with sgRNAs designed to target splice sites to disrupt expression of genes augmenting CAR-T cell therapy, the authors were able to demonstrate efficient multiplex gene editing using base editor technology [57].

In summary, the ability to genetically engineer CAR-T cells carrying receptors for tumour-specific antigens holds great promise for the future of anti-cancer therapy.

More than 90% of human gene variants are caused by single nucleotide variants (SNVs). However, the vast majority of these SNVs lack functional characterisation, and the causal relationship between a variant and disease progression remains unclear.

Previously, assessing this functional relationship between SNVs and phenotype remained technically challenging. Usually performed at small-scale only addressing a few variants at a time to identify their effect on disease progression, high-throughput screening measures remain critical for disease research and cancer genetics.

While a few technologies exist that can screen gene variants in large-scale, for example, saturation mutagenesis, or saturation genome editing (SAGE), they either rely on exogenous overexpression of a variant or inefficient HDR technologies. Therefore, CRISPR-mediated base editor screening offers great promise in overcoming those challenges.

Indeed, recent advancements have been made to establish CRISPR base editors as large-scale screening tools [58–60]. Excitingly, cancer-driving gene variants are a particular focus of base editing screening attempts. Kweon et al. [61] performed a targeted base editing screen to find novel functional variants of the BRCA1 gene. Using the BE3 CBE and 745 sgRNAs targeting all exons of the BRCA1 gene to introduce loss-of-function mutants, they discovered previously unknown variants important for BRCA1 function [61].

In an exciting pre-print publication, Hanna et al. [46] employ large-scale pooled functional screens using cytosine CRISPR base editors. In a first step, they set a benchmark for their base editor screens by validating pathogenic variants of the BRCA1/2 genes. Hanna et al. even went a step further and established base editor screening to identify interaction sites of common chemo-therapeutic drugs, such as BH3 mimetics and PARP inhibitors. This will help identify novel point mutations that render cells resistant to treatment with those drugs. Finally, they created a large library of >68k sgRNAs targeting >50k clinically relevant gene variants via C > T mutation corresponding to around 3500 genes, which identified novel loss-of-function variants required for DNA damage repair and cell stress triggered by low-doses of cisplatin and hygromycin, respectively, in two independent cancer cell lines (HT29, colorectal and MELJUSO, melanoma) [46].

While base editing technology for functional genetic screens is very promising, caution is warranted for the interpretation of results. Gene deletions can be induced by multiple guides targeting different regions of the same gene in both conventional knockout and base editor screens. However, base editor screens used to introduce specific point mutations often rely on the suitability of the guide RNA or cell to cell variability of editing. Therefore, particular importance is placed on validation experiments of primary screens, clearly establishing which edit is causal to the observed phenotype. In the future base editor screens can be further optimised by application of novel Cas variants or base editor architecture, and it will be interesting to see, whether functional genetic screens with ABE editors can recapitulate findings observed with CBE, thereby further strengthening the validation of base editor screens for functional genomic experiments of different gene variants.

Advances in base editor design have greatly improved editing efficiencies [30]. Minimising off-target effects of CBEs remain a critical task to facilitate therapeutic genome editing. While it is beyond the scope of this review to expand on details on the off-target effects associated with base editing experiments, we would like to direct the readers to the landmark report by Rees and Liu [5]. Specific mutations have been made to the Apobec deaminase sequence to reduce random deamination events of RNA and DNA and to improve on-target C-to-T editing [62,63]. In a very promising improvement, the addition of the RAD51 DNA binding domain into the linker sequence between Apobec and Cas9n increases the affinity to bind the exposed single-stranded DNA and therefore improves editing efficiency and reduces editing of neighbouring cytosines (‘Bystander editing’) [64]. In addition, on-target activity remains critical for therapeutic base editing as many SNVs are currently not accessible due to PAM incompatibility. Therefore, advancements to improve the range of CRISPR base editors by widening PAM requirements present a promising avenue [65,66]. In this regard, dual base editors have recently been described capable of introducing both C-to-T and A-to-G mutations, thereby increasing editing potential [67,68]. In addition to those dual base editors, recently Kurt et al. [69] reported the generation of C-to-G base editors (CGBE) capable of introducing transversion mutations into human cells. Recently, attempts have been made to predict base editing outcomes to create disease-associated gene variants [70].

Limitations of current base editor technologies could potentially be overcome by the recently described prime editing technology [71]. Similar to base editors, prime editors are Cas9 fusion proteins, but instead of a deaminase enzyme a nickase version of Cas9 is fused to a reverse transcriptase enzyme which incorporates the desired edit by copying from a template encoded within the designed prime editing guide RNA (pegRNA) [71]. Indeed, prime editing was already successfully used to correct disease-causing mutations in patient-derived intestinal and liver organoids [72]. In another advancement, the first in vivo delivery of RNA-encoded ABE was described to correct a disease-causing mutation in hepatic cells of a tyrosinemia mouse model [73].

Taken together, the ability of genome editing technologies, such as base editing, offer great therapeutic potential to correct disease-causing point mutations.

  • Importance of the field: CRISPR base editing technology offers the unique advantage of introducing precise point mutations without the need of DNA templates. Given the majority of currently know SNVs, including many of the cancer-driving gene variants, remain functionally uncharacterised, base editing tools represent crucial assets for pre-clinical and therapeutic applications.

  • Summary of the current thinking: Since their first development CRISPR base editors are continuously optimised to reduce off-target and increase on-target editing. Base editors show great potential for cancer immunotherapy by multiplex engineering of CAR-T cells.

  • Future directions: Current limitations will be addressed by optimised base editor design aiding the therapeutic potential of this novel genome editing tool. In addition, in vivo base editor screens offer a great advantage for drug discovery to counteract cancer-driving gene variants.

The authors declare that there are no competing interests associated with the manuscript.

M.P. and M.J.H. planned, discussed and wrote the manuscript.

Open access for this article was enabled by the participation of Walter and Eliza Hall Institute in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

We thank all of our current and past colleagues at WEHI. Figures in this manuscript have been generated with images adapted from the Smart Servier Medical Art database (smart.servier.com) under the free CC BY 3.0 license. Our work was supported by grants and fellowships from the Australian National Health and Medical Research Council (NHMRC) (Project Grants 1186575 and 1145728, 1143105, 1159658 and Fellowship 1156095 to M.J.H.), the Leukemia and Lymphoma Society of America (LLS SCOR 7015-18 M.J.H.), the Cancer Council of Victoria (project grant 1147328 and Venture Grant to M.J.H.), the Australian Phenomics Network (to M.P. and M.J.H.). This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council Independent Research Institutes Infrastructure Support Scheme.

     
  • AAVs

    Adeno-associated viruses

  •  
  • ABE

    Adenine base editor

  •  
  • BER

    base excision repair

  •  
  • CAR-T cells

    chimaeric antigen receptor - T cells

  •  
  • CBE

    Cytosine base editor

  •  
  • CGBE

    C-to-G base editors

  •  
  • CRISPR

    clustered regularly interspaced short palindromic repeats

  •  
  • HDR

    homology directed repair

  •  
  • hiPSCs

    human induced pluripotent stem cells

  •  
  • iPSCs

    induced pluripotent stem cells

  •  
  • PAM

    protospacer-adjacent motif

  •  
  • RNP

    ribonucleoprotein

  •  
  • sgRNA

    single-guide RNA

  •  
  • SNVs

    single nucleotide variants

  •  
  • UGI

    uracil glycosylase inhibitor

1
Jinek
,
M.
,
Chylinski
,
K.
,
Fonfara
,
I.
,
Hauer
,
M.
,
Doudna
,
J.A.
and
Charpentier
,
E.
(
2012
)
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity
.
Science
337
,
816
821
2
Sapranauskas
,
R.
,
Gasiunas
,
G.
,
Fremaux
,
C.
,
Barrangou
,
R.
,
Horvath
,
P.
and
Siksnys
,
V.
(
2011
)
The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli
.
Nucleic Acids Res.
39
,
9275
9282
3
Cong
,
L.
,
Ran
,
F.A.
,
Cox
,
D.
,
Lin
,
S.
,
Barretto
,
R.
,
Habib
,
N.
et al (
2013
)
Multiplex genome engineering using CRISPR/Cas systems
.
Science
339
,
819
823
4
Mali
,
P.
,
Yang
,
L.
,
Esvelt
,
K.M.
,
Aach
,
J.
,
Guell
,
M.
,
DiCarlo
,
J.E.
et al (
2013
)
RNA-guided human genome engineering via Cas9
.
Science
339
,
823
826
5
Rees
,
H.A.
and
Liu
,
D.R.
(
2018
)
Base editing: precision chemistry on the genome and transcriptome of living cells
.
Nat. Rev. Genet.
19
,
770
788
6
Lin
,
S.
,
Staahl
,
B.T.
,
Alla
,
R.K.
and
Doudna
,
J.A.
(
2014
)
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery
.
eLife
3
,
e04766
7
Hess
,
G.T.
,
Tycko
,
J.
,
Yao
,
D.
and
Bassik
,
M.C.
(
2017
)
Methods and applications of CRISPR-mediated base editing in eukaryotic genomes
.
Mol. Cell
68
,
26
43
8
Zong
,
Y.
,
Wang
,
Y.
,
Li
,
C.
,
Zhang
,
R.
,
Chen
,
K.
,
Ran
,
Y.
et al (
2017
)
Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion
.
Nat. Biotechnol.
35
,
438
440
9
Shimatani
,
Z.
,
Kashojiya
,
S.
,
Takayama
,
M.
,
Terada
,
R.
,
Arazoe
,
T.
,
Ishii
,
H.
et al (
2017
)
Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion
.
Nat. Biotechnol.
35
,
441
443
10
Qin
,
L.
,
Li
,
J.
,
Wang
,
Q.
,
Xu
,
Z.
,
Sun
,
L.
,
Alariqi
,
M.
et al (
2020
)
High-efficient and precise base editing of C*G to T*A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system
.
Plant. Biotechnol. J.
18
,
45
56
11
Xu
,
Y.
,
Lin
,
Q.
,
Li
,
X.
,
Wang
,
F.
,
Chen
,
Z.
,
Wang
,
J.
et al (
2020
)
Fine-tuning the amylose content of rice by precise base editing of the Wx gene
.
Plant Biotechnol. J.
12
Cai
,
Y.
,
Chen
,
L.
,
Zhang
,
Y.
,
Yuan
,
S.
,
Su
,
Q.
,
Sun
,
S.
et al (
2020
)
Target base editing in soybean using a modified CRISPR/Cas9 system
.
Plant Biotechnol. J.
18
,
1996
1998
13
Qin
,
W.
,
Lu
,
X.
,
Liu
,
Y.
,
Bai
,
H.
,
Li
,
S.
and
Lin
,
S.
(
2018
)
Precise A*T to G*C base editing in the zebrafish genome
.
BMC Biol.
16
,
139
14
Zhang
,
Y.
,
Qin
,
W.
,
Lu
,
X.
,
Xu
,
J.
,
Huang
,
H.
,
Bai
,
H.
et al (
2017
)
Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system
.
Nat. Commun.
8
,
118
15
Yuan
,
H.
,
Yu
,
T.
,
Wang
,
L.
,
Yang
,
L.
,
Zhang
,
Y.
,
Liu
,
H.
et al (
2020
)
Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs
.
Cell Mol. Life Sci.
77
,
719
733
16
Xie
,
J.
,
Ge
,
W.
,
Li
,
N.
,
Liu
,
Q.
,
Chen
,
F.
,
Yang
,
X.
et al (
2019
)
Efficient base editing for multiple genes and loci in pigs using base editors
.
Nat. Commun.
10
,
2852
17
Ma
,
Y.
,
Yu
,
L.
,
Zhang
,
X.
,
Xin
,
C.
,
Huang
,
S.
,
Bai
,
L.
et al (
2018
)
Highly efficient and precise base editing by engineered dCas9-guide tRNA adenosine deaminase in rats
.
Cell Discov.
4
,
39
18
Liu
,
Z.
,
Chen
,
M.
,
Shan
,
H.
,
Chen
,
S.
,
Xu
,
Y.
,
Song
,
Y.
et al (
2019
)
Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7)
.
Cell Mol. Life Sci.
76
,
4155
4164
19
Liu
,
Z.
,
Chen
,
S.
,
Shan
,
H.
,
Jia
,
Y.
,
Chen
,
M.
,
Song
,
Y.
et al (
2020
)
Efficient base editing with high precision in rabbits using YFE-BE4max
.
Cell Death Dis.
11
,
36
20
Liu
,
Z.
,
Shan
,
H.
,
Chen
,
S.
,
Chen
,
M.
,
Song
,
Y.
,
Lai
,
L.
et al (
2020
)
Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits
.
FASEB J.
34
,
588
596
21
Zhou
,
S.
,
Cai
,
B.
,
He
,
C.
,
Wang
,
Y.
,
Ding
,
Q.
,
Liu
,
J.
et al (
2019
)
Programmable base editing of the sheep genome revealed No genome-wide off-target mutations
.
Front. Genet.
10
,
215
22
Zhou
,
S.
,
Ding
,
Y.
,
Liu
,
J.
,
Liu
,
Y.
,
Zhao
,
X.
,
Li
,
G.
et al (
2020
)
Highly efficient generation of sheep with a defined FecB(B) mutation via adenine base editing
.
Genet. Sel. Evol.
52
,
35
23
Zhang
,
W.
,
Aida
,
T.
,
Del Rosario
,
R.C.H.
,
Wilde
,
J.J.
,
Ding
,
C.
,
Zhang
,
X.
et al (
2020
)
Multiplex precise base editing in cynomolgus monkeys
.
Nat. Commun.
11
,
2325
24
Wang
,
F.
,
Zhang
,
W.
,
Yang
,
Q.
,
Kang
,
Y.
,
Fan
,
Y.
,
Wei
,
J.
et al (
2020
)
Generation of a Hutchinson-Gilford progeria syndrome monkey model by base editing
.
Protein Cell
11
,
809
824
25
Komor
,
A.C.
,
Kim
,
Y.B.
,
Packer
,
M.S.
,
Zuris
,
J.A.
and
Liu
,
D.R.
(
2016
)
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
.
Nature
533
,
420
424
26
Mol
,
C.D.
,
Arvai
,
A.S.
,
Sanderson
,
R.J.
,
Slupphaug
,
G.
,
Kavli
,
B.
,
Krokan
,
H.E.
et al (
1995
)
Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA
.
Cell
82
,
701
708
27
Lee
,
K.Y.
,
Lee
,
H.J.
,
Choi
,
H.J.
,
Han
,
S.T.
,
Lee
,
K.H.
,
Park
,
K.J.
et al (
2020
)
Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens
.
FASEB J.
24
,
15907
15921
28
Gaudelli
,
N.M.
,
Komor
,
A.C.
,
Rees
,
H.A.
,
Packer
,
M.S.
,
Badran
,
A.H.
,
Bryson
,
D.I.
et al (
2017
)
Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage
.
Nature
551
,
464
471
29
Surun
,
D.
,
Schneider
,
A.
,
Mircetic
,
J.
,
Neumann
,
K.
,
Lansing
,
F.
,
Paszkowski-Rogacz
,
M.
et al (
2020
)
Efficient generation and correction of mutations in human iPS cells utilizing mRNAs of CRISPR base editors and prime editors
.
Genes (Basel)
11
,
511
30
Anzalone
,
A.V.
,
Koblan
,
L.W.
and
Liu
,
D.R.
(
2020
)
Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors
.
Nat. Biotechnol.
38
,
824
844
31
Merkle
,
F.T.
,
Neuhausser
,
W.M.
,
Santos
,
D.
,
Valen
,
E.
,
Gagnon
,
J.A.
,
Maas
,
K.
et al (
2015
)
Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus
.
Cell Rep.
11
,
875
883
32
Han
,
H.A.
,
Pang
,
J.K.S.
and
Soh
,
B.S.
(
2020
)
Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing
.
J. Mol. Med (Berl).
98
,
615
632
33
Vakulskas
,
C.A.
,
Dever
,
D.P.
,
Rettig
,
G.R.
,
Turk
,
R.
,
Jacobi
,
A.M.
,
Collingwood
,
M.A.
et al (
2018
)
A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells
.
Nat. Med.
24
,
1216
1224
34
Zeng
,
J.
,
Wu
,
Y.
,
Ren
,
C.
,
Bonanno
,
J.
,
Shen
,
A.H.
,
Shea
,
D.
et al (
2020
)
Therapeutic base editing of human hematopoietic stem cells
.
Nat. Med.
26
,
535
541
35
Porto
,
E.M.
,
Komor
,
A.C.
,
Slaymaker
,
I.M.
and
Yeo
,
G.W.
(
2020
)
Base editing: advances and therapeutic opportunities
.
Nat. Rev. Drug Discov.
19
,
839
859
36
Webber
,
B.R.
,
Lonetree
,
C.L.
,
Kluesner
,
M.G.
,
Johnson
,
M.J.
,
Pomeroy
,
E.J.
,
Diers
,
M.D.
et al (
2019
)
Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors
.
Nat. Commun.
10
,
5222
37
Kim
,
K.
,
Ryu
,
S.M.
,
Kim
,
S.T.
,
Baek
,
G.
,
Kim
,
D.
,
Lim
,
K.
et al (
2017
)
Highly efficient RNA-guided base editing in mouse embryos
.
Nat. Biotechnol.
35
,
435
437
38
Li
,
G.
,
Liu
,
Y.
,
Zeng
,
Y.
,
Li
,
J.
,
Wang
,
L.
,
Yang
,
G.
et al (
2017
)
Highly efficient and precise base editing in discarded human tripronuclear embryos
.
Protein Cell
8
,
776
779
39
Xu
,
C.L.
,
Ruan
,
M.Z.C.
,
Mahajan
,
V.B.
and
Tsang
,
S.H.
(
2019
)
Viral delivery systems for CRISPR
.
Viruses
11
,
28
40
Villiger
,
L.
,
Grisch-Chan
,
H.M.
,
Lindsay
,
H.
,
Ringnalda
,
F.
,
Pogliano
,
C.B.
,
Allegri
,
G.
et al (
2018
)
Treatment of a metabolic liver disease by in vivo genome base editing in adult mice
.
Nat. Med.
24
,
1519
1525
41
Yeh
,
W.H.
,
Shubina-Oleinik
,
O.
,
Levy
,
J.M.
,
Pan
,
B.
,
Newby
,
G.A.
,
Wornow
,
M.
et al (
2020
)
In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness
.
Sci. Transl. Med.
12
,
eaay9101
42
Lim
,
C.K.W.
,
Gapinske
,
M.
,
Brooks
,
A.K.
,
Woods
,
W.S.
,
Powell
,
J.E.
,
Zeballos
,
C.M.
et al (
2020
)
Treatment of a mouse model of ALS by in vivo base editing
.
Mol. Ther.
28
,
1177
1189
43
Chen
,
Y.
,
Zhi
,
S.
,
Liu
,
W.
,
Wen
,
J.
,
Hu
,
S.
,
Cao
,
T.
et al (
2020
)
Development of highly efficient dual-AAV split adenosine base editor for in vivo gene therapy
.
Small Methods
4
,
2000309
44
Aubrey
,
B.J.
,
Kelly
,
G.L.
,
Kueh
,
A.J.
,
Brennan
,
M.S.
,
O'Connor
,
L.
,
Milla
,
L.
et al (
2015
)
An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo
.
Cell Rep.
10
,
1422
1432
45
Zafra
,
M.P.
,
Schatoff
,
E.M.
,
Katti
,
A.
,
Foronda
,
M.
,
Breinig
,
M.
,
Schweitzer
,
A.Y.
et al (
2018
)
Optimized base editors enable efficient editing in cells, organoids and mice
.
Nat. Biotechnol.
36
,
888
893
46
Hanna
,
R.E.
,
Hegde
,
M.
,
Fagre
,
C.R.
,
DeWeirdt
,
P.C.
,
Sangree
,
A.K.
,
Szegletes
,
Z.
et al (
2020
)
Massively parallel assessment of human variants with base editor screens
.
bioRxiv
47
Pausch
,
P.
,
Al-Shayeb
,
B.
,
Bisom-Rapp
,
E.
,
Tsuchida
,
C.A.
,
Li
,
Z.
,
Cress
,
B.F.
et al (
2020
)
CRISPR-CasPhi from huge phages is a hypercompact genome editor
.
Science
369
,
333
337
48
Annunziato
,
S.
,
Lutz
,
C.
,
Henneman
,
L.
,
Bhin
,
J.
,
Wong
,
K.
,
Siteur
,
B.
et al (
2020
)
In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer
.
EMBO J.
39
,
e102169
49
Li
,
X.
,
Qian
,
X.
,
Wang
,
B.
,
Xia
,
Y.
,
Zheng
,
Y.
,
Du
,
L.
et al (
2020
)
Programmable base editing of mutated TERT promoter inhibits brain tumour growth
.
Nat. Cell Biol.
22
,
282
288
50
Kueh
,
A.J.
,
Pal
,
M.
,
Tai
,
L.
,
Liao
,
Y.
,
Smyth
,
G.K.
,
Shi
,
W.
et al (
2017
)
An update on using CRISPR/Cas9 in the one-cell stage mouse embryo for generating complex mutant alleles
.
Cell Death Differ.
24
,
1821
1822
51
Lee
,
H.K.
,
Smith
,
H.E.
,
Liu
,
C.
,
Willi
,
M.
and
Hennighausen
,
L.
(
2020
)
Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos
.
Commun. Biol.
3
,
19
52
Zuo
,
E.
,
Sun
,
Y.
,
Wei
,
W.
,
Yuan
,
T.
,
Ying
,
W.
,
Sun
,
H.
et al (
2019
)
Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos
.
Science
364
,
289
292
53
Lee
,
H.K.
,
Willi
,
M.
,
Miller
,
S.M.
,
Kim
,
S.
,
Liu
,
C.
,
Liu
,
D.R.
et al (
2018
)
Targeting fidelity of adenine and cytosine base editors in mouse embryos
.
Nat. Commun.
9
,
4804
54
Cullot
,
G.
,
Boutin
,
J.
,
Toutain
,
J.
,
Prat
,
F.
,
Pennamen
,
P.
,
Rooryck
,
C.
et al (
2019
)
CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations
.
Nat. Commun.
10
,
1136
55
Sharma
,
P.
and
Allison
,
J.P.
(
2015
)
The future of immune checkpoint therapy
.
Science.
348
,
56
61
56
Shi
,
X.
,
Zhang
,
D.
,
Li
,
F.
,
Zhang
,
Z.
,
Wang
,
S.
,
Xuan
,
Y.
et al (
2019
)
Targeting glycosylation of PD-1 to enhance CAR-T cell cytotoxicity
.
J. Hematol. Oncol.
12
,
127
57
Gaudelli
,
N.M.
,
Lam
,
D.K.
,
Rees
,
H.A.
,
Sola-Esteves
,
N.M.
,
Barrera
,
L.A.
,
Born
,
D.A.
et al (
2020
)
Directed evolution of adenine base editors with increased activity and therapeutic application
.
Nat. Biotechnol.
38
,
892
900
58
Kuscu
,
C.
,
Parlak
,
M.
,
Tufan
,
T.
,
Yang
,
J.
,
Szlachta
,
K.
,
Wei
,
X.
et al (
2017
)
CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations
.
Nat. Methods
14
,
710
712
59
Jun
,
S.
,
Lim
,
H.
,
Chun
,
H.
,
Lee
,
J.H.
and
Bang
,
D.
(
2020
)
Single-cell analysis of a mutant library generated using CRISPR-guided deaminase in human melanoma cells
.
Commun. Biol.
3
,
154
60
Despres
,
P.C.
,
Dube
,
A.K.
,
Seki
,
M.
,
Yachie
,
N.
and
Landry
,
C.R.
(
2020
)
Perturbing proteomes at single residue resolution using base editing
.
Nat. Commun.
11
,
1871
61
Kweon
,
J.
,
Jang
,
A.H.
,
Shin
,
H.R.
,
See
,
J.E.
,
Lee
,
W.
,
Lee
,
J.W.
et al (
2020
)
A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants
.
Oncogene
39
,
30
35
62
Koblan
,
L.W.
,
Doman
,
J.L.
,
Wilson
,
C.
,
Levy
,
J.M.
,
Tay
,
T.
,
Newby
,
G.A.
et al (
2018
)
Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction
.
Nat. Biotechnol.
36
,
843
846
63
Gehrke
,
J.M.
,
Cervantes
,
O.
,
Clement
,
M.K.
,
Wu
,
Y.
,
Zeng
,
J.
,
Bauer
,
D.E.
et al (
2018
)
An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities
.
Nat. Biotechnol.
36
,
977
982
64
Zhang
,
X.
,
Chen
,
L.
,
Zhu
,
B.
,
Wang
,
L.
,
Chen
,
C.
,
Hong
,
M.
et al (
2020
)
Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain
.
Nat. Cell Biol.
22
,
740
750
65
Hu
,
J.H.
,
Miller
,
S.M.
,
Geurts
,
M.H.
,
Tang
,
W.
,
Chen
,
L.
,
Sun
,
N.
et al (
2018
)
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
.
Nature
556
,
57
63
66
Huang
,
T.P.
,
Zhao
,
K.T.
,
Miller
,
S.M.
,
Gaudelli
,
N.M.
,
Oakes
,
B.L.
,
Fellmann
,
C.
et al (
2019
)
Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors
.
Nat. Biotechnol.
37
,
626
631
67
Grunewald
,
J.
,
Zhou
,
R.
,
Lareau
,
C.A.
,
Garcia
,
S.P.
,
Iyer
,
S.
,
Miller
,
B.R.
et al (
2020
)
A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing
.
Nat. Biotechnol.
38
,
861
864
68
Zhang
,
X.
,
Zhu
,
B.
,
Chen
,
L.
,
Xie
,
L.
,
Yu
,
W.
,
Wang
,
Y.
et al (
2020
)
Dual base editor catalyzes both cytosine and adenine base conversions in human cells
.
Nat. Biotechnol.
38
,
856
860
69
Kurt
,
I.C.
,
Zhou
,
R.
,
Iyer
,
S.
,
Garcia
,
S.P.
,
Miller
,
B.R.
,
Langner
,
L.M.
et al (
2020
)
CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells
.
Nat. Biotechnol
70
Arbab
,
M.
,
Shen
,
M.W.
,
Mok
,
B.
,
Wilson
,
C.
,
Matuszek
,
Z.
,
Cassa
,
C.A.
et al (
2020
)
Determinants of base editing outcomes from target library analysis and machine learning
.
Cell
182
,
463
480.e30
71
Anzalone
,
A.V.
,
Randolph
,
P.B.
,
Davis
,
J.R.
,
Sousa
,
A.A.
,
Koblan
,
L.W.
,
Levy
,
J.M.
et al (
2019
)
Search-and-replace genome editing without double-strand breaks or donor DNA
.
Nature
576
,
149
157
72
Schene
,
I.F.
,
Joore
,
I.P.
,
Oka
,
R.
,
Mokry
,
M.
,
van Vugt
,
A.H.M.
,
van Boxtel
,
R.
et al (
2020
)
Prime editing for functional repair in patient-derived disease models
.
Nat. Commun.
11
,
5352
73
Jiang
,
T.
,
Henderson
,
J.M.
,
Coote
,
K.
,
Cheng
,
Y.
,
Valley
,
H.C.
,
Zhang
,
X.O.
et al (
2020
)
Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope
.
Nat. Commun.
11
,
1979
74
Levy
,
J.M.
,
Yeh
,
W.H.
,
Pendse
,
N.
,
Davis
,
J.R.
,
Hennessey
,
E.
,
Butcher
,
R.
et al (
2020
)
Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses
.
Nat. Biomed. Eng.
4
,
97
110
75
Ryu
,
S.M.
,
Koo
,
T.
,
Kim
,
K.
,
Lim
,
K.
,
Baek
,
G.
,
Kim
,
S.T.
et al (
2018
)
Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy
.
Nat. Biotechnol.
36
,
536
539
76
Rossidis
,
A.C.
,
Stratigis
,
J.D.
,
Chadwick
,
A.C.
,
Hartman
,
H.A.
,
Ahn
,
N.J.
,
Li
,
H.
et al (
2018
)
In utero CRISPR-mediated therapeutic editing of metabolic genes
.
Nat. Med.
24
,
1513
1518
77
Carreras
,
A.
,
Pane
,
L.S.
,
Nitsch
,
R.
,
Madeyski-Bengtson
,
K.
,
Porritt
,
M.
,
Akcakaya
,
P.
et al (
2019
)
In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model
.
BMC Biol.
17
,
4
78
Yang
,
L.
,
Wang
,
L.
,
Huo
,
Y.
,
Chen
,
X.
,
Yin
,
S.
,
Hu
,
Y.
et al (
2020
)
Amelioration of an inherited metabolic liver disease through creation of a de novo start codon by cytidine base editing
.
Mol. Ther.
28
,
1673
1683
79
Osborn
,
M.J.
,
Newby
,
G.A.
,
McElroy
,
A.N.
,
Knipping
,
F.
,
Nielsen
,
S.C.
,
Riddle
,
M.J.
et al (
2020
)
Base editor correction of COL7A1 in recessive dystrophic epidermolysis bullosa patient-derived fibroblasts and iPSCs
.
J. Invest. Dermatol.
140
,
338
47.e5
80
Song
,
C.Q.
,
Jiang
,
T.
,
Richter
,
M.
,
Rhym
,
L.H.
,
Koblan
,
L.W.
,
Zafra
,
M.P.
et al (
2020
)
Adenine base editing in an adult mouse model of tyrosinaemia
.
Nat. Biomed. Eng.
4
,
125
130
81
Geurts
,
M.H.
,
de Poel
,
E.
,
Amatngalim
,
G.D.
,
Oka
,
R.
,
Meijers
,
F.M.
,
Kruisselbrink
,
E.
et al (
2020
)
crispr-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank
.
Cell Stem Cell
26
,
503
510.e7
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