Cytosine case, high-throughput DNA sequencing (HTS) was

Cytosine Base Editors (BE1, BE2, BE3 BE4) For thepurpose of precise single base editing, a number of plasmids called baseeditors (BE) were developed during 2016-17. These plasmids facilitate baseediting (a transition) involving conversion of cytosine into uracil (Fig. 3),leading to replacement of cytosine/guanine (C:G) base pair by thymine/adenine(T:A) base pair. Since these base editors were meant for alteration of cytosineonly, these could be better named as cytosine base editors (CBE) as againstadenine base editors (ABE) that were developed for A®I(G) conversion later in 2017 (I = inosine).     The first-generation C®U base editors (BE1) were developed using the rat cytidinedeaminase AID/APOBEC1 connected to a disabled Cas9 (dCas9) via a 16 base XTENlinker4 (Komor et al. 2016).

AID/APOBECs (activationinduced deaminase/ apolipoprotein B mRNA editing enzyme, catalyticpolypeptide-like) used in this study represent a family of naturally occurringcytidine deaminases, which use single-stranded DNA/RNA as a substrate11(Knisbacher et al. 2016). Themembers of AID/APOBEC family were combined with the CRISPR/dCas9 system toperform targeted base editing. This combination improved CRISPR/Cas9-mediatedgene editing at single base precision, thus greatly enhancing its utility. Theoriginal requirements for single base editing included the followingcomponents: (i) a disabled Cas9 (dCas9) fused to a cytidine deaminase; (ii) a gRNA that helps dCas9 to target a specific locus associated witha protospacer adjacent motif (PAM) sequence available ~18-20 base pairs downstream, and (iii) a target cytosine within a window of positions 4-8. These firstgeneration base editors (BE1) were further improved leading to the developmentof a series of base editors that were described as second generation, thirdgeneration and fourth generation base editors12 (BE2, BE3, BE4)(Table 1). In each case, high-throughput DNA sequencing (HTS) was used to quantify baseediting efficiency. Digenome seq (sequencing of digested DNA) was also used forassessment of off-target effects in human cells13 (Kim, D et al.

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2015). Improvement of BEsusing Uracil N-Glycolase Inhibitors (UGI) The major problem withthe first generation base editors (BE1) included the formation of undesired productsdue to the following two reasons: (i) frequent removal of uracil by cellular N-glycosylase(UNG) and (ii) possible occurrence of more than one Cs within the base editingactivity window of 4-8 bases, permitting base editing of non-target cytosines possible.The enzyme UNG worksduring Base Excision Repair (BER) and therefore, will identify transitional editedbase pair G:U as DNA damage and will excise U in G:U base pair, which is usedfor the conversion of G:C into T:A base pair. Keeping this in view and in orderto increase in vivo editingefficiency, second generation base editors (BE2) were developed, which carrieda uracil glycosylase inhibitor (UGI) fused with dCas9, so that the enzyme UNGwill not be able to excise U from the G:U base pair. The editing efficiency of thesesecond-generation base editors (BE2) was three-fold that of BE1 reaching amaximum of ~20%; indel formation was very low (<0.1%) both in BE1 and  BE2, since the DNA was not directly cleavedas in case of CRTISPR-mediated genome editing.

The second problem of theoccurrence of more than one Cs in the editing window was partly resolved byreducing the size of editing window to 1 or 2 base pairs (see later).      The next stage ofimprovement of base editors was achieved by converting dCas9 to a nickase throughreplacement of either amino acid aspartate (D) by alanine (A) atposition 10 (D10A; also described as Cas9n), or replacement of amino acidhistidine (H) by alanine at position 840 (H840A). Cas9n and H840A both produce nicks inopposite strands, and have been suitably utilized in single base gene editing14(Ran et al., 2013). For instance D10A mutant of Cas9 retains a domain thatgenerates a single strand DNA nick in the non-target strand instead ofcreating double strand breaks at the desired site; this would simulate mismatchrepair, so that a unmodified opposite DNA strand would mimic a DNA strandundergoing synthesis, where the strand containing the edited base is used as a template(C ®U; Fig. 4), takingU as T. Therefore, BE3 had the following three components: (i) an AID/APOBEC1 deaminase,that was fused through a 16–amino acid linker to (ii) a Streptococcus pyogenes nickase Cas9n Cas9n(D10A), whichwas first disabled for its nuclease function and was later converted into anickase (Cas9n) and (iii) a UGI that was linked to Cas9n through a 4 aminoacids linker. The importance of UGI in base editing was demonstrated by showing thatthe UGI-deleted BE3 (BE3-?UGI ) was less competent in base editing compared to originalBE3, and produced not only lower frequency of desired C®T editing,but also produced a higher frequency of unwanted indels.

A number of improvedBE3 variants were also developed (Table 2), which resulted in much moreefficient conversion of the G:U intermediate to desired A:U and A:T products4,11(Komor et al., 2016, 2017).     Another problem associated with BE1 and BE2 was the occurrence of morethan one Cs within the base editing activity window, so that the cytosinedeaminase will convert even a non-targeted C into U. This problem was overcome by thedevelopment of BE3 with SpCas9 (NGG), where even the non-NGG PAM sequence couldbe used for base editing.     It was also shown that addition of anothercopy of UGI to BE3 further reduced the frequency of indels, so that BEs werelater improved by having more than one copy of UGI associated with Cas9n andcytosine deaminase. These were described as fourth generation base editors, theBE4, which were found to be more efficient (Wang et al., 2017).  BE4 or SaBE4 were further improved by addingGam to the cassette, so that the use of BE4-Gam resulted in a further 1.

5 to2.0 fold decrease in the indel frequency (Table 1). Table 2. BE3 variants developed by Kim et al. (2017) and Rees et al.

(2017)Fig. 5. PAM sequence 18 bases downstream of the site for basealteration; the base editing occurs in arange of few bases surrounding the –18 position upstream of PAM sequence of thenon-target DNA strand. PAM site and Efficiency of BaseEditors (Kim et al., 2017) Ithas been recognized that Cas9 will not bind and cleave the target DNA sequence successfully,if the target is not followed by a 2-6 base pair long protospacer adjacentmotif (PAM) sequence, that lies immediately following the DNA sequence targetedby the Cas9 using guide RNA (gRNA). Although gRNAs can transport Cas9 to anyspecific sequence in the genome for gene editing, but no editing can occur if PAMsequence is absent. The canonical PAM is 5′-NGG-3′ that is associated with theCas9 nuclease of Streptococcus pyogenes(designated SpCas9), whereas different PAMs are associated with the Cas9proteins of other bacteria like Neisseriameningitidis, Treponema denticola,and Streptococcus thermophilus.

Forinstance, the PAM sequence 5′-NGA-3′ can be a highly efficient non-canonicalPAM for human cells, but efficiency varies with genome location. Attempts have been made toengineer Cas9s to recognize different PAMs to improve ability of CRISPR/Cas9 todo gene editing at any desired genome location.   Fig. 6. A schematic for the plasmid BE4-Gam, carrying APOBEC1(cytidine deaminase), Cas9 (D10A) and two copies of UGI (uracil N-glycosylaseinhibitor)       Efficient editing by BE3 requires the presence of an NGGPAM that places the target C within a five-nucleotide window near thePAM-distal end (positions 4–8, counting the PAM as positions 21–23, with a 16nt linker; Komor et al.

20164). This PAM requirementsubstantially limits the number of sites in the human genome that can beefficiently targeted by BE3, because many sites of interest lack an NGG 13- to17- nucleotides downstream of the target C. Moreover, the high activity of BE3results in conversion of all Cs to Us within the editing window, which canpotentially introduce undesired changes to the target locus. Improved editorsaddress both of these limitations (lack of NGG PM and editing non target Cs)and thereby substantially expand the targets suitable for base editing.      The new fourth generation base editors(BE4s) would expand the number of targetable loci by allowing non-NGG PAM sitesto be edited. Also, the Cas9 homolog from Staphylococcusaureus (SaCas9) is considerably smaller than SpCas9 (1,053 vs.

1,368residues); it has been successfully used for efficient genome editing inmammalian cells, and requires an NNGRRT PAM. For instance, a BE3 nickase form ofSaCas9 was successfully used with HEK293T cells (kidney cells) for targetingsix human genomic loci (Fig. 1a). A very high frequency of C to U(T) conversionwas reported in these studies14 (…….

).      Two other important papers were publishedin 2017 demonstrating significant improvement in the efficiency of BE3 editors.Kim et al. (2017)15 modified Cas9 variants with specific anddistinct PAM sequences (for expanding the number of available target sites forbase editing), and mutagenized cytidine deaminase to create SpCas9 base editorswith editing windows as small as 1-2 nucleotides. The minimum editingefficiency was improved to ~50%. Rees et al. (2017)16 reported developmentof high fidelity base editors (HF-BE3), which contained high fidelity Cas9variant HF-Cas9, leading to 37-fold reduction in off-target editing, with onlya slight reduction in on-target editing efficiency.

They also purified HF-BE3protein for delivery in the form of ribonucleoprotein particles (RNPs) to bothzebrafish embryos and the mouse inner ear.     Fourth-generation base editors (BE4 andSaBE4) were developed through the addition of another copy of UGI resulting in(i) increased efficiency of C:G to T:A base editing by approximately 50%, and(ii) reducing the frequency of undesired by-products to half relative to thatin base editing via BE3.  Improvement in BE4base editors was also achieved by fusion of Phage Mu Gam protein that bindsDSBs, leading to reduction in indel formation to <1.5%, andimprovement in product purity. In this manner several BE4 vectors including BE4,SaBE4, BE4-Gam, and SaBE4-Gam became available, which represented the state ofthe art in C:G-to-T:A base editing17 (Komor et al.

, 2017).Different components of a BE4-Gam plasmid vector are shown in Figure 4.         Fig. 7. Schematic diagram illustrating the design of sgRNA, associatedwith BE3, eBE-S1 and eBE-S3 expression vectors                                                                                                                                      Fig. 8.

A schematic representation of cytosine base editing; cytosine deamination takesplace in the first strand converting C to U, without producing a DSB. BE3 nicksthe opposite strand, to help mismatch repair, thus retaining the edited base. Enhanced BEs(eBE-S1, eBE-S3) and High Fidelity Bes (HF-BE Two other classes of improvedBEs that were developed included enhanced base editors (eBEs) and high fidelityBEs (HF-BEs). An enhanced BE (eBE) involved designing of a vector that would co-expressBE3 with either one (eBE-S1) or three (eBE-S3) copies of 2A-UGI sequence(Figure 5). The approach has since been deployed in a range of organisms,from wheat to zebrafish and mice.

 Fig. 9.Schematic of the high-fidelity base editor (HF-BE3) described in Rees et al.bound to target DNA. HF-BE3 contains HF-Cas9 nickase, a uracil DNA glycosylaseinhibitor (UGI) and a cytidine deaminase. Image from Rees H, et al.

2017.High fidelity BEs involved modification of Cas9 through 4-5mutations. High-fidelity 1 variant (Cas9-HF1) contained four point mutations(N497A/R661A/Q695A/Q926A), while Cas9 high-fidelity 2 (Cas9-HF2) contained oneadditional mutation (D1135E) compared to Cas9-HF1.

HF2-BE2 exhibited alteredPAM preference (from NGG/A to NGG only), and exhibited higher level ofspecificity18,19 (Kleinstiver et al., 2016;Kleinstiver et al., 2015). High fidelity HF-HF3 were also developed, which had reduced BE3off-target activity20 (Rees et al.

(2017) These HF-BEs (HF1-BE1 andHF-BE2) were shown to successfully convert C to T with up to 100% efficiency inmouse embryos21 (Liang et al., 2017). Adenine BaseEditors (ABEs)Initially, base editing was restricted to C®T conversion involving use of naturally occurring DNA cytosinedeaminase. However no DNA adenine deaminase occurs in nature, although RNAadenine deaminase occur for modification of tRNAs, so that synthetic DNAadenine deaminases had to be developed from RNA adenine deaminase throughprotein engineering using directed evolution.  Following synthesis of DNAadenine deaminase in the laboratory, base editors could be developed forconversion of adenine into inosine (I) (Fig. 10); the latter mimics guanine (G)during DNA replication, so that T:A base pair could be edited  into C:G base pair  (Fig.

8). Table 3. BaseEditing Publication Highlights Publication Plasmids (Base Editors) Highlights Komor et al. 2016 BE1, BE2, BE3 BE3 displays highest editing efficiency but higher indel formation than BE2 Nishida et al. 2016 Target-AID Edits 3-5 base window surrounding -18 position upstream of the PAM Kim et al. 2017 SaB®3, BE3 PAM variants, BE3 editing window variants Greatly expands the number of target loci for base editing Rees et al.

2017 HF-BE3 HF-BE3 and ribonucleoprotein delivery decrease BE3 off-target activity Komor et al. 2017 BE4 and BE4-Gam; AID, CDA1 and APOBEC3G BE3 variants A second copy of UGI improves product purity.  Gam decreases indel frequency. Gaudelli et al. 2017 Adenine base editors (ABE) A®I (A®G) editing with high product purity and low off-target editin The newsystem of base editors for A®Gconversion were called Adenine Base Editor (ABE). As in case of other baseeditors, ABEs also consisted of a guide RNA, an adenine deaminase and amodified form of CRISPR­Cas9.

The development of novel DNA adenine deaminasesin the laboratory ultimately resulted in high­performance, seventh generationABEs, which included the following four ABEs: ABE 6.3, ABE 7.8, ABE 7.9 andABE 7.109 (Gaudelli et al.,2017).

Of these, ABE7.10 was the most active editor, displaying an averageediting efficiency of 53% with an editing window of target positions 4-7. Theother three ABEs displayed slightly wider editing windows of position 4-9,although editing efficiency was lower at positions 8 and 9.

It was alsoobserved that ABEs do not display significant A to non-G conversion at targetloci, since the removal of inosine from DNA is not as common as that of uracil(U)9 (Gaudelli et al. (2017). ABEs also performed better than manyBEs in terms of off-target editing and frequency of indels produced duringediting. In an actual study, ABE7.10 modified only 4/12 off-targets with afrequency of 1.2% indels, in comparison with 9/12 known off-targets with a 14%indel rate in other BEs.

       Base Editing in RNA In most studies onbase editing, DNA is used as the target, but similar approach can be used forRNA also. Cox et al. (2017)21 reported successful editing of adenineinto inosine in RNA molecule. This allows for a temporary correction of adisease-causing mutation without permanent alteration in the genome.  Fig. 12. A newly created adenine base editor(ABE) with the following components: (i) an adenine deaminase (red), which canchange adenine into inosine (equivalent to guanine); (ii) guide RNA or gRNA(green), which directs the molecule to the right spot as in CRISPR technology,and (iii) Cas9 nickase (blue), which snips the opposing strand of DNA andtricks the cell into swapping the complimentary base (after Gaudelli et al.,2017)9.

 A comparison of RNA base editing with CRISPRand DNA base editing is shown in Figure 10.  Fig. 13. A comparison ofCRISPR-mediated genome editing, DNA base editing and RNA base editing 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 bediscussed. (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 to sickle-cellanemia to metabolic disorders to cystic fibrosis, for which no treatments areavailable 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 andpredictable genetic change for treatment of a diseases.

Using mouse cells grownin culture, it has been shown that the mutations associated withAlzheimer’s disease can be corrected using BEs with an efficiency of up to 75%.Similarly, using human cells, mutation in a gene associated with a cancer couldbe corrected with up to 7.6% efficiency. These corrections could not bepossible using standard CRISPR­Cas9 method.

Therefore, “base editors” and notCRISPR/Cas9, can correct many harmful point mutations that are associated witha number of diseases, for which no treatments are available at present. In order to make it a reality for human health care, deliveryof this machine and its safety and side effects are the questions, which arebeing 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, Zong et al. (2017)used two plant base editors (PBE) carrying CASPR-nCas9 -cytidine deaminase(APOBEC1) fusion proteins, namely nCas9-PBE and dCas9-PBE (Fig…). These weresuccessfully used for base editing in rice, wheat and maize with frequencies ofindividual cytosine editing ranging from 5% to 32.5%, with no  associated indels.

  (iii) In maize….(iv) In tomato,…..