Targeted dual base editing with Campylobacter jejuni Cas9 by a single AAV-mediated delivery

doi:10.21203/rs.3.rs-1973343/v1

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Targeted dual base editing with Campylobacter jejuni Cas9 by a single AAV-mediated delivery

https://doi.org/10.21203/rs.3.rs-1973343/v1

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published 01 Feb, 2023

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Various CRISPR-Cas9 orthologues are used in genome engineering. One of the smallest Cas9 orthologues is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPDgene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffold can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.

CRISPR-Cas

Genome editing

base editing

AAV delivery

CRISPR-Cas9 is a powerful tool for genome engineering, and Streptococcus pyogenes Cas9 (spCas9) is the most widely used Cas9 for genome editing in various living systems. However, the large size of spCas9 protein poses a barrier for in vivo or ex vivo delivery (1). Aside from spCas9, many other Cas9 orthologs and their guide RNAs (gRNAs) have been identified (2–7), of which Campylobacter jejuni Cas9 (cjCas9) is one of the smallest Cas9 orthologs. cjCas9 consists of 984 amino-acid residues, which is significantly less than the 1,384 amino-acids residues of spCas9 (8). cjCas9 has several distinct characteristics compared with spCas9; for example, cjCas9 recognizes a 5’-NNNNRYAC-3’ PAM sequence, whereas spCas9 recognizes a 5’-NGG-3’ PAM sequence. Crystal structure analysis showed that cjCas9 can interact with both the target and non-target strands of the PAM sequence, while spCas9 recognizes the PAM sequence through a non-target strand (9). In addition, cjCas9 induces indel mutations in human cells with longer gRNAs compared with those of spCas9, as the length of gRNAs for cjCas9 and spCas9 are 22-nt and 20-nt, respectively (8).

Recently, several groups developed cjCas9-based genome engineering toolkits. Zhang et al. generated cjCas9-based transcription activators (termed miniCAFE) and showed that miniCAFE can activate target genes in human cells and Caenorhabditis elegans (10). Zhang et al. also showed that miniCAFE can activate an endogenous gene in vivo through an all-in-one adeno-associated virus (AAV) delivery. Because of the small size of cjCas9, cjCas9 has an advantage over spCas9, especially in an AAV delivery system.

CRISPR-mediated base editors (BEs), which have deaminase enzyme fused to Cas9-nickase, are powerful tools for functional assessment of point mutations, which represent the largest class of pathogenic mutations (11). To date, cytidine base editors (CBEs) and adenine base editors (ABEs) have been developed (12–14), and improved versions of CBEs and ABEs have been reported to have enhanced activity and specificity (15–19). Recent studies showed that cjCas9 can also be used as BEs. Li et al. generated cjABE by fusing cjCas9-D8A with Escherichia coli (E.coli)-derived evolved-tRNA adenine deaminase (Tad A, ABE7.10 version), and successfully corrected TERT promoter mutation by AAV infection (20). Nakagawa et al. developed cjCas9-AID by fusing cjCas9-D8A with Petromyzon marinus cytosine deaminase 1 (PmCDA1) and showed that engineered cjCas9 (encjCas9, L58Y/D900K variants)-based cjCBE can induce targeted base editing in human cells, whereas wild-type cjCas9 based cjCBE cannot introduce endogenous base substitutions (21). Although the studies by Li et al. and Nakagawa et al. showed that cjCas9 could be used as BEs, their characteristics were not analyzed in detail and further studies are needed to define their functional characterization.

In this study, we developed cjCBEmax (cjCas9-D8A fused with APOBEC1 from BE4max) that induces a C:G to T:A conversion, and cjABE8e (cjCas9-D8A fused with evolved-TadA from ABE8e) that induces an A:T to G:C conversion. Using cjCBEmax and cjABE8e, we could induce base substitutions at 13 endogenous target sites with high frequency and characterize their active windows and context-dependency. Subsequently, we also applied encjCas9 and engineered gRNA scaffold (e-scaffold) to improve base editing efficiencies, and successfully constructed an all-in-one AAV vector containing cjABE8e and dual gRNAs and induced base substitutions by AAV delivery.

Plasmids constructs

pRGEN-CMV-CjCas9 (#89752; addgene, Watertown, MA, USA) was used for pCMV-cjCas9 transfection and pU6-Cj-sgRNA (addgene #89753) was used to generate the gRNAs of cjCas9. The target sequences of gRNAs are provided in Supplementary Table 1. pCMV-cjCas9-D8A was generated by inducing a point mutation in the pRGEN-CMV-CjCas9 vector. The engineered E.coli-tRNA adenosine deaminase domain was amplified from ABE8e (addgene #138489) and cloned into pCMV-cjCas9-D8A to generate a pCMV-cjABE8e vector. The APOBEC1 cytidine deaminase domain and UGI domains from pCMV-BE4MAX-3xHA (addgene #112096) were amplified and cloned into pCMV-cjCas9-D8A to generate pCMV-cjCBEmax. pAAV-EFS-cjCas9-eEGFP-HIFIa (addgene #137929) was modified to make pAAV-cjABE8e-ANGPT2-HPD-2. The constructs of pCMV-cjABE8e, pCMV-cjCBEmax, and pAAV-cjABE8e-2xU6 (gRNA empty vector of AAV vectors) will be available in addgene.

Cell culture and analysis of mutation frequency

HEK293T (ATCC CRL-3216) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10 % fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ℃ in a 0.05% CO₂ atmosphere. Mycoplasma detection tests were performed every 2 weeks to confirm the absence of mycoplasma contamination. One day before transfection, 2.5 ´ 10⁴ HEK293T cells were seeded onto 96-multiwell plates, and transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, 250 ng of plasmids (125 ng of gRNAs, 125 ng of pCMV-cjCas9 variants) were transfected in 60% confluent HEK293T cells and genome DNA was extracted using cell lysis buffer (0.05% SDS in pH 7.5 of 100 nM Tris-HCl and 100 μg/ml proteinase K) 72 h after transfection. The cell lysate was stored at -20 ℃ until use. To measure the mutation frequencies of each sample, genomic DNA was amplified with target-specific primer pairs listed in Supplementary Table 1, and NGS libraries were further generated with TruSeq HT Dual Index primer pairs. The NGS libraries were subjected to paired-end sequencing using Mini-seq or iSEQ (Illumina, San Diego, CA, USA) and data were analyzed using MAUND as previously described ²².

Production and titration of AAV vector

HEK293T cells were transfected with pAAV-cjABE8e-gRNA-ANGPT2-HPD-2, pAAV-DJ encoding for AAV2rep, AAV-DJcap, and helper plasmids and maintained in DMEM with 2% FBS. Recombinant pseudotyped AAV vector stock was generated by PEI coprecipitation using PEI-MAX (Polysciences, Warrington, PA, USA) and transfection was conducted with three plasmids at a molar ratio of 1:1:1. After 72 h of incubation, the cells were lysed and AAV particles were purified by iodixanol step-gradient ultracentrifugation. The number of vector genomes was determined by quantitative PCR.

AAV transduction in cells

HEK293T cells were infected with AAV particles at a multiplicity of different viral genome (VG)/cell ranging from 1 ´ 10² vg/cell, 1 ´ 10³ vg/cell, and 1 ´ 10⁴ vg/cell determined by quantitative PCR and maintained in DMEM with 2% FBS. After 72 h of incubation, the infected cells were harvested and subjected to targeted deep sequencing.

Western blot assay

Three plasmids—pRGEN-CMV-CjCas9, pCMV-cjABE8e, and pCMV-cjCBEmax—were transfected in HEK293T cells, and whole-cell lysates (WCLs) were prepared using 1 ´ RIPA lysis buffer 72 h after transfection and quantified by Bradford assay. Western blot assay was conducted using the iWestern system (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, 10 μg of WCLs were separated in 4%–12% bis-tris gradient gels and transferred to a polyvinylidene difluoride membrane using the iBlot 2 Dry Blotting System. Immunoblotting was conducted with anti-HA (#9110; Abcam, Cambridge, UK) and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH, sc-47724; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. The immunoblotted proteins were detected by enhanced chemiluminescence.

Statistics and Reproducibility

All experiments were performed in biologically independent triplicates, and the data are reported as mean ± standard error of the mean and plotted using GraphPad Prism (GraphPad Software, La Jolla, CA, USA).

Construction of cjCBEmax and cjABE8e

We used improved variants of spCas9-based BEs—BE4MAX and ABE8e—to generate highly active cjCas9-based BEs—cjCBEmax and cjABE8e. The two improved base editors (BE4MAX and ABE8e) were developed and reported in the literature ^15,19; BE4MAX contains codon-optimized APOBEC1 cytidine deaminase and UGI domains, and the ABE8e has an evolved TadA deoxyadenosine deaminase. We cloned these domains into the pCMV-cjCas9-D8A construct to generate cjCBEmax and cjABE8e (Figure 1A). Compared with the original spCas9-based BEs, the coding sequences of cjCBEmax and cjABE8e were approximately 1.3-fold smaller and their expression levels were confirmed by Western Blot assay (Supplementary Figure 1). We transfected these constructs into HEK293T cells with AAVS1-2 and AAVS1-8 gRNA, which was shown to allow cjCas9 to induce indel with a high mutation frequency ⁸, and analyzed the mutation frequencies by targeted deep sequencing.

The conventional active window of base editors was 4–9 positions in their spacer sequences; however, because we did not know the active window of the cjCas9-based BEs, we analyzed base editing frequencies over a broader range, including 15-nt outside the spacer sequence. At the AAVS1-2 and AAVS1-8 target sites, cjABE8e induced A:T to G:C conversions by up to 55.7% and 49.3%, and the cjCBEmax introduced C:G to T:A conversions by up to 18.1% and 19.1%, respectively (Figure 1B). Interestingly, both cjCBEmax and cjABE8e were able to introduce base substitutions outside the conventional active windows (Figures 1C and 1D). cjABE8e induced an A:T to G:C conversion by up to 32.0% at the A(15) position of AAVS1-2 target sites and 43.0% at the A(11) position of AAVS1-8 target sites.

Although there was no cytosine in the conventional active window, cjCBEmax could induce base C:G to T:A conversions in both AAVS1-2 and AAVS1-8 target sites, especially by up to 11.3% at the C(-9) position of the AAVS1-8 target site. At these two sites, we found that cjABE8e could not efficiently edit adenines located outside the spacer sequence, whereas cjCBEmax could convert cytosines located outside the spacer sequence. Taken together, we demonstrated that cjCBEmax and cjABE8e can induce base conversion at endogenous target sites, suggesting that their active window might be much wider than that of conventional BEs.

Characterization of cjCBEmax and cjABE8e

To further characterize cjCBEmax and cjABE8e, we cloned gRNAs to edit 11 additional target sites and transfected them with cjCas9, cjCBEmax, and cjABE8e in HEK293T cells. As shown in Figure 2A, cjCBEmax induced C:G to T:A conversions by up to 43.5% at the HPD-1 target site and cjABE8e induced A:T to G:C conversions by up to 54.3% at the HPD-2 target site. We found that cjABE8e had higher activity than cjCBEmax in all target sites, with cjABE8e and cjCBEmax showing average mutation frequencies of 35.4% and 17.4%, respectively, across 13 endogenous target sites. To characterize the base editing active window of cjCBEmax and cjABE8e, we analyzed the substitution frequencies of individual cytosines and adenines in a 50-nt window (Figure 2B and Supplementary Figure 2). In line with the results on AAVS1-2 and AAVS1-8 (Figures 1C and 1D), we found that cjCBEmax and cjABE8e had a wider active window compared with spCas9-based BEs. cjCBEmax could edit the C(-12) position by up to 1.9% in the EPAS1-1 target site and the C(19) position by up to 0.7% in the HIF1A-1 target site. Particularly in the SERPINC1 target site, cjCBEmax induced base substitutions by up to 13.5% at the C(13) position, which was a much higher frequency than those of the C(8) located in the conventional active window. Compared with cjCBEmax, cjABE8e showed a narrower active window across the 13 target sites, inducing base substitutions by up to 9.4% at the A(-3) position of the ANGPT2 target site and 7.1% at the A(18) position of the EPAS1-1 target site.

Next, we examined whether the context around adenines and cytosines affected the base editing activities of cjCBEmax and cjABE8e. For cjABE8e, the AA sequence context had an adverse effect on the adenine base editing activity, while the TA sequence context tended to enhance the base editing activity (Figure 2C). The cjCBEmax exhibited a relatively high cytosine editing activity in the context of a TC sequence compared with that in the context of a GC sequence (Figure 2D). Previously, Song et al. analyzed the correlation between the efficiency of spCas9-based BEs and sequence context ²³, and we found that cjCBEmax and cjABE8e showed similar trends to the spCas9-based BEs. Overall, we demonstrated that cjCBEmax and cjABE8e can induce substitutions at various endogenous sites with a wider active window than that of spCas9-based BEs, and that the sequence context affecting their activity was similar to that of the spCas9-based BEs.

Specificity of cjCBEmax and cjABE8e

Next, we assessed the tolerance of cjCBEmax and cjABE8e for mismatched gRNAs. A total of 22 gRNAs having one or two mismatches with the target sequences were constructed and transfected in HEK293T cells with cjCas9, cjCBEmax, and cjABE8e (Supplementary Table 1). We analyzed the indel and base substitution frequencies by targeted deep sequencing and compared the tolerance for mismatched gRNAs (Figure 2E). In most cases, cjCBEmax and cjABE8e were tolerant to base mismatches in the PAM-distal region than in the PAM-proximal region, whereas HPD-2-M1-1 and HPD-2-M1-2 mismatched gRNAs showed a different trend. For example, cjCBEmax showed 44.2% base editing activity with HPD-2-M1-2-mismatched gRNA containing a 1-bp mismatch at position 20 of the spacer sequence, which was comparable to the base editing activity with HPD-2 gRNA. As the mismatch tolerance for HPD-2-M1-1 and HPD-2-M1-2, which had a 1-bp mismatch at the closest location in the PAM, was also observed with cjCas9, we speculated that this might be a characteristic of cjCas9 or a target-specific trait.

For most gRNAs with 2-bp mismatches, cjCas9 was not tolerant, whereas cjCBEmax and cjABE8e had modest tolerance that was proportional to the distance from the PAM region. Especially with HPD-2-M2-11-mismatched gRNA containing 2-bp mismatches at positions 1 and 2, cjCBEmax and cjABE8e induced substitutions by up to 15.0% and 35.6%, respectively, whereas cjCas9 showed an indel frequency of 0.9%. These results suggest that cjCBEmax and cjABE8e have a slightly lower specificity compared with cjCas9.

We also sought to identify the endogenous off-target effects of cjCBEmax and cjABE8e. The potential off-target sites of each gRNAs were analyzed in silico using Cas-OFFinder ²⁴, and we selected an AAVS1-8-OT1 potential off-target site containing 2-bp mismatched to their target spacer sequence (Supplementary Table 2). We analyzed the endogenous mutations by targeted deep sequencing and found that cjCBEmax and cjABE8e as well as cjCas9 showed no detectable endogenous off-target mutations (Figure 2F).

Improvement of cjCBEmax and cjABE8e

To improve the base editing efficiency of cjCBEmax and cjABE8e, we engineered the scaffold sequences of gRNAs according to previous study regarding to the spCas9 gRNA engineering. As shown in Figure 3A, we removed a putative terminator motif of four consecutive uracils by single A:U to G:C conversion to avoid premature termination of gRNA transcription and truncated the tetraloop to shorten the length of gRNAs and named the engineered scaffold as “e-scaffold”. We cloned gRNAs with e-scaffold for five endogenous target sites and compared their mutation frequencies to those of wild-type scaffold by targeted deep sequencing. As shown in Figure 3B, the e-scaffold improved the mutation frequencies of cjCas9, cjCBEmax, and cjABE8e at all five target sites. Especially at the EPAS1-2 target site, gRNAs with e-scaffold enhanced the base editing frequencies of cjCBEmax by 3.1-fold.

A recent study showed that an L58Y/D900K double mutation in cjCas9 (encjCas9) can improve the activity of cjCas9 ²¹. To determine whether the L58Y/D900K double mutation was synergetic with e-scaffold, we first compared the indel frequency of cjCas9 and encjCas9 combinations with gRNAs with the wild-type scaffold or e-scaffold (Figure 3C). We found that encjCas9 had improved activity compared with cjCas9 and had synergetic effects with e-scaffold across five target sites; especially, at the HIF1A-2 target site, the combination of encjCas9 and e-scaffold enhanced the indel activities by 7.6-fold (from 4.0% to 30.3%). We then introduced the L58Y/D900K double mutation in cjABE8e (encjABE8e) and tested their activity with gRNAs bearing e-scaffold (Figure 3D). In five target sites, encjABE8e showed improved base editing activity compared with cjABE8e, which were synergetic with e-scaffold.

AAV vector of cjABE8e for base editing

We next examined whether the cjABE8e could be packaged into an AAV vector. Because of the limited packaging capacity, spCas9-based BEs have challenge to be delivered through a single AAV vector systems . Since cjABE8e was small enough to package into an AAV vector, we speculated that cjABE8e and two tandem arrays of gRNA might be integrated into a single AAV vector (Figure 4A). To further reduce the size of the construct, we investigated whether a previously known synthetic polyadenylation (polyA) sequence was compatible with cjABE8e. As the synthetic polyA sequence is 49-bp long, which is much shorter than the 225-bp bovine growth hormone (BGH) polyA sequence, it can provide more space for AAV packaging.

We first cloned single-pAAV-cjABE8e constructs containing BGH polyA or synthetic polyA sequence and transfected them in HEK293T cells (Figure 4B). Targeted deep sequencing showed that single-pAAV-cjABE8e constructs with synthetic polyA and BGH polyA sequence had similar base editing frequencies across five target sites. We then constructed a dual-pAAV-cjABE8e vector containing two gRNAs targeting ANGPT2 and HPD-1 and compared its base editing efficiency with those of single-pAAV-cjABE8e vectors. As a result, we found that dual-pAAV-cjABE8e vector could induce base substitutions by 68.4% and 82.9% at the ANGPT2 and HPD-1 targe sites, respectively, which were comparable with those of single-pAAV-cjABE8e vectors (Figure 4C). Subsequently, we produced AAV particles and infected them into HEK293T cells and found that the base editing frequencies were accumulated in a dose-dependent manner up to 24.0% and 91.9% at the ANGPT2 and HPD-1 target sites, respectively (Figure 4D). We also investigated potential off-target sites of ANGPT2 and HPD-1 in silico and measured mutations at these sites by targeted deep sequencing in AAV-infected HEK293T cells, but did not find detectable off-target mutations (Figure 4E and Supplementary Table 2).

In this study, we demonstrated that cjCas9-based BEs cjCBEmax and cjABE8e can induce nucleotide substitutions in the human genome with high efficiency and that their active window is much wider than those of spCas9-based BEs. We further improved the base editing activity of cjCBEmax and cjABE8e by applying engineered cjCas9 and a scaffold of gRNAs. Due to their small size, cjABE8e and two tandem arrays of gRNAs can successfully be packaged into a single-AAV vector, a powerful tool for efficient genome editing in vivo.

CRISPR-meditated BEs have emerged as powerful tools for therapeutic uses in human diseases, but in vivo delivery of BEs remains a major challenge due to the large size of BEs (1). Recently, several groups showed that ABEs containing smaller Cas9 orthologues than spCas9, Staphylococcus aureus Cas9 (saCas9), Staphylococcus Auricularis Cas9 (SauriCas9) and Neisseria meningitidis Cas9 (Nme2Cas9) with their single gRNA can be embedded into a single-AAV vector (25, 26). Compared with saCas9- and Nme2Cas9-ABE, cjABE8e is smaller in size so there is more room for packaging other constructs in a single-AAV vector such as one additional copy of gRNA. Using this construct, we successfully introduced base substitutions in multiple loci by single AAV delivery.

We found that cjCas9-based BEs have wider active windows than spCas9-based Bes, with cjABE8e being able to edit an adenine at positions (-3) to (+ 18) and cjCBEmax being able to edit a cytosine at positions (-6) to (+ 18). Especially, cjCas9-BEs can introduce base substitutions outside the target spacer sequences, which is likely because cjCas9 is small enough for deaminases to approach the target DNA. Same as cjCas9-BEs, saCas9-BEs have a wider active window than spCas9-BEs (27, 28). The active window of cjCas9-based BEs can be made narrower for target-specific nucleotide substitution; however, the feature of a wide active window may be an advantage for use in gene silencing, including disruption of coding sequences and canonical splice sites without inducing double-strand breaks in the DNA (29–33).

Recent studies by Li et al. (20, 21) and Nakagawa et al. (20, 21) reported the generation of cjCas9-AID, cjCas9-D8A nickase fused with PmCDA1, and cjCas9-based ABE (termed cjABE), cjCas8-D8A nickase fused with TadA (ABE7.10 version). Although these studies showed that cjCas9 could be used as BEs, their characteristics, including active window and context-dependency, were not analyzed in detail. Nakagawa et al. showed that the cjCas9-AID mostly failed to induce C:G to T:A conversions and they developed encjCas9-AID, encjCas9-D8A nickase fused with AID, for targeted cytidine base editing. The encjCas9-AID, but not cjCas9-AID, showed base editing activity in human cells, whereas cjCBEmax containing wild-type cjCas9 showed high base editing activity in this study. Li et al. generated cjABE and corrected − 124 C > T TERT promoter mutation via AAV delivery in vivo. Compared with cjABE, cjABE8e used in this study has a much smaller size enough to be packaged with dual gRNAs in AAV vectors and showed higher base editing activity.

In summary, we successfully developed small-sized base editors, cjCBEmax and cjABE8e, using cjCas9 and defined their functional characteristics including active windows and context-dependency. We expect that these cjCas9-mediated base editors capable of delivery with a single AAV can be used to broaden the usage of base editors in biomedical research.

DATA AVAILABILITY

The sequencing data for this study are available from the Sequencing Read Archive (https://www.ncbi.nlm.nih. gov/sra) under accession numbers PRJNA850677

SUPPLEMENTARY DATA

Supplementary Data are available at EMM online.

ACKNOWLEDGEMENT

We thank members of the Center for Genome Engineering, Institute for Basic Science for their assistance and resources for high-throughput sequencing. We thank the Scientific Publications Team at Asan Medical Center for editorial assistance in preparing the paper.

FUNDING

This work was supported by the National Research Foundation of Korea [2017M3A9B4062419, 2020R1F1A1075508, 2018R1A5A2020732 to Y.K. ; 2022R1A2C1013352 to T.K.].

CONFLICT OF INTEREST

The authors declare no competing financial interests.

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Targeted dual base editing with Campylobacter jejuni Cas9 by a single AAV-mediated delivery

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