Applications of Base Editing A numberof BEs and ABEs have been used for a wide variety of applications, includingplant genome editing, in vivomammalian genome editing, targeted mutagenesis, and knockout studies (1, 7–9, 12–19).Some of these applications including future possibilities will be discussed. (a) Application of Base Editing in Human Health As shownin this article, the base editors including BEs and ABEs can correct each ofthe the following four “transition” mutations; C®T, T®C, A® G, or G® A, which together account for almost two-thirds of alldisease-causing point mutations. Many of these mutations, each involving singlebase alteration cause serious diseases, ranging from genetic blindness tosickle-cell anemia to metabolic disorders to cystic fibrosis, for which no treatmentsare available at present. It has been estimated that approximately half of the32,000 disease-associated point mutations already identified by researchers area change from G:C to A:T, which can be corrected by BE3, BE4 and theirdifferent variants. These are also diseases, which involve changes from A:T toG:C, which can be corrected using ABEs. These base editors couldhelp in the future development of gene-therapy approaches (Gaudelli et al.
,2017)9. Additional research is, however, needed to enable BEs and ABEs totarget as much of the genome as possible. The BEs and ABEs developed by David Liu andhis team have been described as molecular machines, which make the desired and predictablegenetic change for treatment of a diseases. Using mouse cells grown in culture,it has been shown that the mutations associated with Alzheimer’s disease can becorrected using BEs with an efficiency of up to 75%. Similarly, using humancells, mutation in a gene associated with a cancer could be corrected with upto 7.6% efficiency. These corrections could not be possible using standardCRISPRCas9 method.
Therefore,”base editors” and not CRISPR/Cas9, can correct many harmful point mutationsthat are associated with a number of diseases, for which no treatments areavailable at present. In order to make it a realityfor human health care, delivery of this machine and its safety and side effectsare the questions, which are being addressed. Researchers in China also reportedthat they had used Liu’s base editor to correct a single base mutation or pointmutation, for a blood disorder in human embryos (The embryos were not allowedto develop further). A team in Korea usedmouse zygotes as a model system and targeted the Dmd or Tyr gene.F0 mice showed nonsensemutations with an efficiency of 44–57% and allelic frequencies of up to 100%,demonstrating an efficient method to generate mice with targeted pointmutations (Kim et al., 2017).
(2) Application ofBase Editing in Crop Improvement Examples ofsuccessful base editing are also available in plants. In most cases, a BE3variant with nCas9 nickase fused with a cytidine deaminase and a UGI was used forbase editing. Since delivery of template DNA can sometimes be a problem inplants, a target-AID (target-activation-inducedcytidine deaminase) was used as cytidine deaminase (Shimatani et al., 2016), and the fusion product was codon optimized for plants(cereals); these base editors were, therefore, described as plant base editor =PBE (Zong et al.
(2017). Thecrops, which were used for base editing included cereals (rice, wheat ad maize (Zonget al., 2017), rice and tomato (Shimataniet al., 2017) These examples include the following: (i) Ina study in rice, nCas9 nickase was fused with a cytidine deaminase enzyme and a UGIto generate targeted mutations.
The BE3 cassette was inserted in pCXUN vectorto generate pCXUN-BE3, which had the ability to target a specified locus, whena gRNA molecule is simultaneously expressed (….). Expression cassette of a gRNAunder the control of the rice U3 promoter was inserted into the PmeIsite of pCXUN-BE3. Three targets were chosen: one target (P2)in the OsSBEIIb gene,which encodes a phytoene desaturase, and two targets (S3 and S5) in the gene OsSB,which encodes a starch branching enzyme IIb. The vectors were delivered intorice calli through Agrobacterium- mediated transformation. Thebase-editing vectors demonstrated their feasibility and efficacy (Li et al.,2017).
Base editing was successful at all the three loci with efficiency muchhigher than obtained using CRISP/Cas9 system. Zong etal. (2017) (ii) In another study, Zonget al. (2017) used two plant base editors (PBE) carrying CASPR-nCas9 -cytidinedeaminase (APOBEC1) fusion proteins, namely nCas9-PBE and dCas9-PBE (Fig…).These were successfully used for base editing in rice, wheat and maize withfrequencies of individual cytosine editing ranging from 5% to 32.
5%, with no associated indels. (iii) In maize….(iv) In tomato,…..