Base editing has pounced onto the gene-editing stage. Base editing presents a useful alternative to HDR-mediated gene editing with active Cas9 nuclease, avoiding DSBs and the resulting indels. With base editors, scientists can convert one base to another, and this nucleotide conversion happens without double-stranded cutting by fully active CRISPR-Cas9 nuclease, without invoking DNA repair mechanisms that follow double-stranded breaks or using a donor template. Base editing systems make use of Cas9 variants, cytidine deaminases, and manipulation of DNA repair pathways to achieve speciﬁc editing outcomes. Capable of generating either precise C>T edits or targeted diversiﬁcation of a genomic region, base editing systems have been applied to therapeutic correction of deleterious mutations, engineering of agricultural crops, and the study and evolution of protein structure and function.
Base editing will also help scientists build more accurate cancer models to expand on information from large-scale tumor sequencing. Many cancer mutations are highly recurrent single-nucleotide changes and some genes have hot-spot mutations waiting to be studied in detail. Base editing is an easier way to deliver editing machinery to cells, and some evidence suggests base editing makes fewer unwanted insertions and deletions than classic CRISPR-Cas9 system. As with any emerging technology, base editing systems will require additional ﬁne tuning and characterization, especially for therapeutic applications of precise base editing. Nonetheless, base editing is an exciting new addition to the CRISPR-related toolbox, and its potential in genome engineering and biology is just beginning to be realized.
Base editing involves site-speciﬁc modiﬁcation of the DNA base along with manipulation of the DNA repair machinery to avoid faithful repair of the modiﬁed base. Base editors are chimeric proteins composed of a DNA targeting module and a catalytic domain capable of deaminating a cytidine or adenine base. There is no need to generate DSBs to edit DNA bases, thereby limiting the generation of insertions and deletions (indels) at target/off-target sites. In general, base editing systems fall into two principle types: precision edits including BE3, YEE-BE3, target AID and SaBE4-Gam; diversifying edits including TAM and CRISPR-X. The different base editing systems own different architectures, catalytic activities, and potential modiﬁcations. In most such systems, DNA targeting module is based on dCas9 guided by an sgRNA molecule. Cas9 nickase can also be used as the targeting module, resulting in high frequencies of base editing.
Fig 1. The mechanism of base editing technologies.
Fig 3. Screening applications of base editing.
Base editors come in a number of flavors. The first generation of base editors uses cytidine deaminase APOBEC1 (RNA-editing enzyme) fused to catalytically impaired Cas9 (dCas9), in which the two nuclease domains of Cas9 are inactivated. Transfection of BE1 and a targeted sgRNA successfully converted cytidines to thymidines within a window of -16 to -12 bases from the PAM of the sgRNA in the first initial BE1 system. An optimized BE2 system resulted from the addition of a base-edit repair inhibitor, the uracil DNA glycosylase inhibitor (UGI) from the Bacillus subtilis bacteriophage PBS1. UGI directly binds and inhibits uracil DNA glycosylase, thus blocking uridine excision and the ensuing BER pathway. This improvement increased base editing efﬁciency of the C>T substitution 3-fold by effectively disfavoring error-free repair.
The optimized BE3 editor consists of rAPOBEC1 fused to the N terminus of nickase Cas9 D10A and a UGI fused to the C terminus. This iteration, BE3, achieved a 6-fold increase in efﬁciency over BE2, with up to 37% of targeted alleles edited. Fourth-generation base editors, BE4, involve modifications such as an additional copy of a repair inhibitor. Scientists also engineered adenine base editors (ABEs), which in their view are the most useful ones in that they can convert A·T to G·C, and thus can revert the most common mutation in living systems. For base editors, the mechanisms of DNA repair are critical for the eventual editing outcome. After the initial deamination (C to U) conversion, uracil DNA glycosylase can remove the base and perform error-free (correctly replacing U with C) or error-prone repair, generating different substitutions.
Fig 2. Part of base editing systems.
Though base editing technologies are still new, there have already been many successful applications in vitro and in vivo, ranging from precise therapeutic and agronomic editing to broad screens for directed evolution and mapping of protein-drug interactions. Base editors provide effective reagents to potentially treat human genetic diseases, two-thirds of which are due to single-base alterations. Moreover, such base editors can help model, study, and correct various genetic diseases. For example, work with human embryos is also under way. One research group altered a mutation that causes β-thalassemia using cloned human embryos made from a patient's cells. A separate team achieved, "genetic correction" in human embryos heterozygous for a mutation underlying Marfan syndrome. As with CRISPR-Cas9, ethics discussions are likely to accompany developments in base editing.
Therefore, the most important use of base editors is in gene therapy, and to treat debilitating genetic diseases. However, there are many potential applications across eukaryotic systems for a variety of basic biology and biotechnology purposes. For example, studies have demonstrated the applications of base editors in developing herbicide resistance. Indeed, targeted mutagenesis has been used to edit the ALS gene to develop herbicide resistance. Precise base editing has been successfully implemented in wheat, rice, maize, and tomato. Important applications examining protein functions and producing functional variants are expected to revolutionize basic research and biotechnological applications. Base editing hold great promise for applications in trait development in crops and these efforts would accelerate trait development and expand the range of traits in agriculture. In addition, The precise base editing system can also be used for screening applications.
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