Cytosine was used to quantify base editing efficiency. Digenome

Cytosine Base Editors (BE1, BE2, BE3 BE4)

 

For the
purpose of precise single base editing, a number of plasmids called base
editors (BE) were developed during 2016-17. These plasmids facilitate base
editing (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 cytosine
only, these could be better named as cytosine base editors (CBE) as against
adenine base editors (ABE) that were developed for A®I(G) conversion later in 2017 (I = inosine).

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     The first-generation C®U base editors (BE1) were developed using the rat cytidine
deaminase AID/APOBEC1 connected to a disabled Cas9 (dCas9) via a 16 base XTEN
linker4 (Komor et al. 2016).
AID/APOBECs (activation
induced deaminase/ apolipoprotein B mRNA editing enzyme, catalytic
polypeptide-like) used in this study represent a family of naturally occurring
cytidine deaminases, which use single-stranded DNA/RNA as a substrate11
(Knisbacher et al. 2016). The
members of AID/APOBEC family were combined with the CRISPR/dCas9 system to
perform targeted base editing. This combination improved CRISPR/Cas9-mediated
gene editing at single base precision, thus greatly enhancing its utility. The
original requirements for single base editing included the following
components: (i) a disabled Cas9 (dCas9) fused to a cytidine deaminase; (ii) a gRNA that helps dCas9 to target a specific locus associated with
a protospacer adjacent motif (PAM) sequence available ~18-20 base pairs downstream, and (iii) a target cytosine within a window of positions 4-8. These first
generation base editors (BE1) were further improved leading to the development
of a series of base editors that were described as second generation, third
generation and fourth generation base editors12 (BE2, BE3, BE4)
(Table 1). In each case, high-throughput DNA sequencing (HTS) was used to quantify base
editing efficiency. Digenome seq (sequencing of digested DNA) was also used for
assessment of off-target effects in human cells13 (Kim, D et al.
2015).

 

Improvement of BEs
using Uracil N-Glycolase Inhibitors (UGI)

 

The major problem with
the first generation base editors (BE1) included the formation of undesired products
due 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 editing
activity window of 4-8 bases, permitting base editing of non-target cytosines possible.
The enzyme UNG works
during Base Excision Repair (BER) and therefore, will identify transitional edited
base pair G:U as DNA damage and will excise U in G:U base pair, which is used
for the conversion of G:C into T:A base pair. Keeping this in view and in order
to increase in vivo editing
efficiency, second generation base editors (BE2) were developed, which carried
a uracil glycosylase inhibitor (UGI) fused with dCas9, so that the enzyme UNG
will not be able to excise U from the G:U base pair. The editing efficiency of these
second-generation base editors (BE2) was three-fold that of BE1 reaching a
maximum of ~20%; indel formation was very low (<0.1%) both in BE1 and  BE2, since the DNA was not directly cleaved as in case of CRTISPR-mediated genome editing. The second problem of the occurrence of more than one Cs in the editing window was partly resolved by reducing the size of editing window to 1 or 2 base pairs (see later).      The next stage of improvement of base editors was achieved by converting dCas9 to a nickase through replacement of either amino acid aspartate (D) by alanine (A) at position 10 (D10A; also described as Cas9n), or replacement of amino acid histidine (H) by alanine at position 840 (H840A). Cas9n and H840A both produce nicks in opposite strands, and have been suitably utilized in single base gene editing14 (Ran et al., 2013). For instance D10A mutant of Cas9 retains a domain that generates a single strand DNA nick in the non-target strand instead of creating double strand breaks at the desired site; this would simulate mismatch repair, so that a unmodified opposite DNA strand would mimic a DNA strand undergoing synthesis, where the strand containing the edited base is used as a template (C ®U; Fig. 4), taking U 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), which was first disabled for its nuclease function and was later converted into a nickase (Cas9n) and (iii) a UGI that was linked to Cas9n through a 4 amino acids linker. The importance of UGI in base editing was demonstrated by showing that the UGI-deleted BE3 (BE3-?UGI ) was less competent in base editing compared to original BE3, and produced not only lower frequency of desired C®T editing, but also produced a higher frequency of unwanted indels. A number of improved BE3 variants were also developed (Table 2), which resulted in much more efficient 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 more than one Cs within the base editing activity window, so that the cytosine deaminase will convert even a non-targeted C into U. This problem was overcome by the development of BE3 with SpCas9 (NGG), where even the non-NGG PAM sequence could be used for base editing.      It was also shown that addition of another copy of UGI to BE3 further reduced the frequency of indels, so that BEs were later improved by having more than one copy of UGI associated with Cas9n and cytosine deaminase. These were described as fourth generation base editors, the BE4, which were found to be more efficient (Wang et al., 2017).  BE4 or SaBE4 were further improved by adding Gam to the cassette, so that the use of BE4-Gam resulted in a further 1.5 to 2.0 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 base alteration; the base editing occurs in a range of few bases surrounding the –18 position upstream of PAM sequence of the non-target DNA strand.   PAM site and Efficiency of Base Editors (Kim et al., 2017)   It has 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 adjacent motif (PAM) sequence, that lies immediately following the DNA sequence targeted by the Cas9 using guide RNA (gRNA). Although gRNAs can transport Cas9 to any specific sequence in the genome for gene editing, but no editing can occur if PAM sequence is absent. The canonical PAM is 5'-NGG-3' that is associated with the Cas9 nuclease of Streptococcus pyogenes (designated SpCas9), whereas different PAMs are associated with the Cas9 proteins of other bacteria like Neisseria meningitidis, Treponema denticola, and Streptococcus thermophilus. For instance, the PAM sequence 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells, but efficiency varies with genome location. Attempts have been made to engineer Cas9s to recognize different PAMs to improve ability of CRISPR/Cas9 to do 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-glycosylase inhibitor)         Efficient editing by BE3 requires the presence of an NGG PAM that places the target C within a five-nucleotide window near the PAM-distal end (positions 4–8, counting the PAM as positions 21–23, with a 16 nt linker; Komor et al. 20164). This PAM requirement substantially limits the number of sites in the human genome that can be efficiently targeted by BE3, because many sites of interest lack an NGG 13- to 17- nucleotides downstream of the target C. Moreover, the high activity of BE3 results in conversion of all Cs to Us within the editing window, which can potentially introduce undesired changes to the target locus. Improved editors address 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 sites to be edited. Also, the Cas9 homolog from Staphylococcus aureus (SaCas9) is considerably smaller than SpCas9 (1,053 vs. 1,368 residues); it has been successfully used for efficient genome editing in mammalian cells, and requires an NNGRRT PAM. For instance, a BE3 nickase form of SaCas9 was successfully used with HEK293T cells (kidney cells) for targeting six human genomic loci (Fig. 1a). A very high frequency of C to U(T) conversion was reported in these studies14 (…….).      Two other important papers were published in 2017 demonstrating significant improvement in the efficiency of BE3 editors. Kim et al. (2017)15 modified Cas9 variants with specific and distinct PAM sequences (for expanding the number of available target sites for base editing), and mutagenized cytidine deaminase to create SpCas9 base editors with editing windows as small as 1-2 nucleotides. The minimum editing efficiency was improved to ~50%. Rees et al. (2017)16 reported development of high fidelity base editors (HF-BE3), which contained high fidelity Cas9 variant HF-Cas9, leading to 37-fold reduction in off-target editing, with only a slight reduction in on-target editing efficiency. They also purified HF-BE3 protein for delivery in the form of ribonucleoprotein particles (RNPs) to both zebrafish embryos and the mouse inner ear.      Fourth-generation base editors (BE4 and SaBE4) 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 that in base editing via BE3.  Improvement in BE4 base editors was also achieved by fusion of Phage Mu Gam protein that binds DSBs, leading to reduction in indel formation to <1.5%, and improvement in product purity. In this manner several BE4 vectors including BE4, SaBE4, BE4-Gam, and SaBE4-Gam became available, which represented the state of the 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, associated with BE3, eBE-S1 and eBE-S3 expression vectors                                                                                                                                       Fig. 8. A schematic representation of cytosine base editing; cytosine deamination takes place in the first strand converting C to U, without producing a DSB. BE3 nicks the 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 improved BEs that were developed included enhanced base editors (eBEs) and high fidelity BEs (HF-BEs). An enhanced BE (eBE) involved designing of a vector that would co-express BE3 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 glycosylase inhibitor (UGI) and a cytidine deaminase. Image from Rees H, et al. 2017. High fidelity BEs involved modification of Cas9 through 4-5 mutations. High-fidelity 1 variant (Cas9-HF1) contained four point mutations (N497A/R661A/Q695A/Q926A), while Cas9 high-fidelity 2 (Cas9-HF2) contained one additional mutation (D1135E) compared to Cas9-HF1. HF2-BE2 exhibited altered PAM preference (from NGG/A to NGG only), and exhibited higher level of specificity18,19 (Kleinstiver et al., 2016; Kleinstiver et al., 2015). High fidelity HF-HF3 were also developed, which had reduced BE3 off-target activity20 (Rees et al. (2017) These HF-BEs (HF1-BE1 and HF-BE2) were shown to successfully convert C to T with up to 100% efficiency in mouse embryos21 (Liang et al., 2017).   Adenine Base Editors (ABEs) Initially, base editing was restricted to C®T conversion involving use of naturally occurring DNA cytosine deaminase. However no DNA adenine deaminase occurs in nature, although RNA adenine deaminase occur for modification of tRNAs, so that synthetic DNA adenine deaminases had to be developed from RNA adenine deaminase through protein engineering using directed evolution.  Following synthesis of DNA adenine deaminase in the laboratory, base editors could be developed for conversion 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. Base Editing 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 new system of base editors for A®G conversion were called Adenine Base Editor (ABE). As in case of other base editors, ABEs also consisted of a guide RNA, an adenine deaminase and a modified form of CRISPR­Cas9. The development of novel DNA adenine deaminases in the laboratory ultimately resulted in high­performance, seventh generation ABEs, which included the following four ABEs: ABE 6.3, ABE 7.8, ABE 7.9 and ABE 7.109 (Gaudelli et al., 2017). Of these, ABE7.10 was the most active editor, displaying an average editing efficiency of 53% with an editing window of target positions 4-7. The other three ABEs displayed slightly wider editing windows of position 4-9, although editing efficiency was lower at positions 8 and 9. It was also observed that ABEs do not display significant A to non-G conversion at target loci, 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 many BEs in terms of off-target editing and frequency of indels produced during editing. In an actual study, ABE7.10 modified only 4/12 off-targets with a frequency 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 on base editing, DNA is used as the target, but similar approach can be used for RNA also. Cox et al. (2017)21 reported successful editing of adenine into inosine in RNA molecule. This allows for a temporary correction of a disease-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 can change 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 and tricks the cell into swapping the complimentary base (after Gaudelli et al., 2017)9.  A comparison of RNA base editing with CRISPR and DNA base editing is shown in Figure 10.     Fig. 13. A comparison of CRISPR-mediated genome editing, DNA base editing and RNA base editing Applications of Base Editing   A number of BEs and ABEs have been used for a wide variety of applications, including plant genome editing, in vivo mammalian 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 shown in this article, the base editors including BEs and ABEs can correct each of the the following four "transition" mutations; C®T, T®C, A® G, or G® A, which together account for almost two-thirds of all disease-causing point mutations. Many of these mutations, each involving single base alteration cause serious diseases, ranging from genetic blindness to sickle-cell anemia to metabolic disorders to cystic fibrosis, for which no treatments are available at present. It has been estimated that approximately half of the 32,000 disease-associated point mutations already identified by researchers are a change from G:C to A:T, which can be corrected by BE3, BE4 and their different variants. These are also diseases, which involve changes from A:T to G:C, which can be corrected using ABEs. These base editors could help in the future development of gene-therapy approaches (Gaudelli et al., 2017)9. Additional research is, however, needed to enable BEs and ABEs to target as much of the genome as possible.      The BEs and ABEs developed by David Liu and his team have been described as molecular machines, which make the desired and predictable genetic 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 be corrected using BEs with an efficiency of up to 75%. Similarly, using human cells, mutation in a gene associated with a cancer could be corrected with up to 7.6% efficiency. These corrections could not be possible using standard CRISPR­Cas9 method. Therefore, "base editors" and not CRISPR/Cas9, can correct many harmful point mutations that are associated with a number of diseases, for which no treatments are available at present. In order to make it a reality for human health care, delivery of this machine and its safety and side effects are the questions, which are being addressed.    Researchers in China also reported that they had used Liu's base editor to correct a single base mutation or point mutation, for a blood disorder in human embryos (The embryos were not allowed to develop further).      A team in Korea used mouse zygotes as a model system and  targeted the Dmd or Tyr gene. F0 mice showed nonsense mutations with an efficiency of 44–57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations (Kim et al., 2017). (2) Application of Base Editing in Crop Improvement   Examples of successful base editing are also available in plants. In most cases, a BE3 variant with nCas9 nickase fused with a cytidine deaminase and a UGI was used for base editing. Since delivery of template DNA can sometimes be a problem in plants, a target-AID (target-activation-induced cytidine 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).      The crops, which were used for base editing included cereals (rice, wheat ad maize (Zong et al., 2017), rice and tomato (Shimatani et al., 2017)        These examples include the following: (i) In a study in rice, nCas9 nickase was fused with a cytidine deaminase enzyme and a UGI to generate targeted mutations. The BE3 cassette was inserted in pCXUN vector to generate pCXUN-BE3, which had the ability to target a specified locus, when a gRNA molecule is simultaneously expressed (….). Expression cassette of a gRNA under the control of the rice U3 promoter was inserted into the PmeI site 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 into rice calli through Agrobacterium- mediated transformation. The base-editing vectors demonstrated their feasibility and efficacy (Li et al., 2017). Base editing was successful at all the three loci with efficiency much higher than obtained using CRISP/Cas9 system.   Zong et al. (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 were successfully used for base editing in rice, wheat and maize with frequencies of individual cytosine editing ranging from 5% to 32.5%, with no  associated indels.  (iii) In maize….(iv) In tomato,…..