1. Introduction
With continuous advancements in gene editing technology, CRISPR/Cas9 has emerged as one of the most extensively employed tools in gene editing [
1,
2]. However, safety concerns associated with this tool have gradually heightened in bio-pharmaceutical applications [
3,
4].The activation of Cas9 protein for the cleavage of the target DNA is contingent upon the presence of a protospacer adjacent motif (PAM) sequence on the sgRNA-matched target DNA sequence. The utilization of a single sgRNA to direct Cas9 in recognizing the target site for genetic modification is frequently constrained by RNA–DNA mismatches, and the conventional Cas9 induces some off-target effects, particularly a close similarity of 10 base pairs following the sgRNA sequence [
5,
6,
7]. To mitigate off-target effects, researchers have developed various high-fidelity Cas9 variants such as HFCas9 [
8], eCas9 [
9], HypaCas9 [
10], or other variants. These variants involve the mutations of amino acids within the catalytic region or the DNA binding pocket of the Cas9 protein, aiming to impose stricter catalytic conditions, such as tighter DNA binding, thereby increasing the specificity of gene editing. However, these Cas9 variants reportedly exhibit favorable editing activity, albeit with varying degrees of reduced editing efficiency [
11,
12,
13]. The reduction in targeting efficiency may be attributed to structural modifications or the weakened DNA binding ability of the Cas9 enzyme.
Furthermore, a strategy known as “dual sgRNAs” could be employed to diminish off-target effects. This strategy necessitates the correct positioning of both left and right sgRNAs for DNA cleavage to occur, thereby enhancing editing specificity. This approach has been employed in two implementation strategies. One involves two sgRNAs guiding nCas9 for cutting the target region [
14], while the other includes FokI endonuclease-fused dCas9 (fdCas9) [
15,
16]. In the latter case, dCas9 is responsible for searching the target sequence, and cleavage is executed by the dimerized FokI enzyme. Before the study of fdCas9, editing tools like TALEN and ZFN utilized dimerized FokI for gene editing [
17]. Importantly, dCas9 could not cleave the genome, and off-target effects from fdCas9 theoretically occurred only when both sgRNAs had mismatches in proximity. When utilizing fdCas9 for gene targeting, DNA cleavage can only occur when both sgRNAs guide the FokI molecules into the correct positions, and the dimerized FokI is able to cleave the gene. However, the available target sites are significantly limited due to the requirement of a protospacer adjacent motif (PAM) sequence. Specifically, the presence of an ′NGG′ PAM at both ends of the targeting sequence is necessary.
In recent years, efforts to expand the repertoire of PAM motifs have led to the engineering of various Cas9 variants. The emergence of gene nucleases, such as XCas9, has broadened the spectrum of target sites [
18]. Notably, RYCas9 [
19], a specific variant, showcases a remarkable nearly PAM-less feature, enabling it to cleave diverse genomic sequences. RYCas9 demonstrates higher editing efficiency with an NRN (R = A/G) PAM compared to NYN (Y = C/T). However, the efficacy of editing through the fusion of the FokI enzyme and deactivated RYCas9 remains unknown, necessitating further evaluation to assess both on-target and off-target effects.
In this study, we successfully established a novel gene-editing tool, termed fRYdCas9, by combining the FokI endonuclease with the nearly PAM-less RYdCas9 variant. This tool presented a much more flexible PAM demand than the reported high-fidelity Cas9 version, while demonstrating precise editing characteristics. We have confirmed that fRYdCas9 exhibits a 330-fold increase in available targeting loci compared to fdCas9 across the entire human genome. Additionally, our findings demonstrate that fRYdCas9 displays comparable editing efficacy to fdCas9. Notably, optimal editing efficiency for fRYdCas9 was observed with a spacer length of 16 base pairs. Importantly, utilizing library screening and whole-genome sequencing (WGS), we discovered that fRYdCas9 demonstrates a remarkable ability to recognize target sites without strict PAM requirements and without showing any preference for specific motifs. This characteristic significantly reduces the occurrence of sg-dependent off-target effects. We also confirmed the fRYdCas9 has robust editing capabilities in mouse embryos. In conclusion, our study indicates the high-fidelity and efficient DNA editing potential of fRYdCas9. It could provide an additional, safer choice for future gene therapy or other applications, especially in cases where other high-fidelity Cas9 variants are unable to perform editing at the desired target site.
2. Materials and Methods
2.1. Plasmid Construction
The Cas9 construct was based on the PX330 plasmid, generously provided by Feng Zhang (Addgene plasmid #42230). CMV and mCherry were inserted into the Cas9 plasmid. RYCas9 construction followed literature protocols, and dCas9 and dRYCas9 were obtained by introducing two-point mutations, D10A and H840A. Subsequently, FokI was inserted into them to generate fdCas9 and fRYdCas9, provided by David Liu (in addition to gene plasmid #42230). For sgRNA expression plasmids, sgRNA oligos were annealed and inserted before the scaffold (cloned from PX330) by T4 ligations. All plasmids were extracted following the E.Z.N.A plasmid kit manuals.
For library constructions, oligonucleotides were synthesized, and PCRs were performed (
Table S6). The library’s oligonucleotides were cloned into a MluI- and XbaI-digested backbone of the lentiviral plasmid. The Gibson assembly method was employed for library construction. After transduction for 20 h, all DH5α colonies were collected, and plasmids were extracted.
2.2. Cell Culture, Transfection, and Genotyping
HEK293T and K562 cells were cultured in DMEM (high glucose) medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Meilunbio, Dalian, China). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO
2. For transfection, we co-transfected 2 ug of the editor plasmid and 1 ug sgRNA into HEK293T cells using poly-ethyleneimine (PEI, Polyscience, Warrington, PA, USA) following the manufacturer’s protocols in 12-well plates. Positive cells were isolated by flow cytometry 48 h after transfection, and genomic DNA was extracted using the Crude DNA Extraction Kit (catalog number P072, Vazyme, Nanjing, China). The genotyping of transfected cells was determined using gene-specific primers through nested PCR. In the first round of PCR amplification, Extaq (Takara, Shiga, Japan) was activated at 95 °C for 3 min, then for 30 cycles, with denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, with an extension at 72 °C for 1 min and a final extension at 72 °C for 5 min after the cycles. The second round of PCR was performed with inner nested primers through the same PCR program. The PCR products were purified for Sanger sequencing or next-generation sequencing. The genotyping identification primers used in this study are listed (
Table S7). Next-generation sequencing data were analyzed using CRISPResso2 (version 2.2.7).
2.3. Lentiviral Vector Production and Transduction
For lentiviral production, we collected supernatants containing lentiviral particles 48 h after transfecting HEK293T cells with 30 μg library lentiviral vector, 22.5 μg of psPAX2, and 15 μg of pMD2.G in a 15 cm dish. For the lentiviral transduction of HEK293T cells, the library cell lines were incubated with the lentiviral supernatant. The HEK293T cells, stably expressed in the library, were generated through lentiviral transduction at an MOI of 0.3. This was followed by the selection of EGFP-positive cells using FACS sorting. The library cells were then transfected with editor and sgRNA vectors, and double-positive cells (mCherry+ EGFP+) were sorted out by FACS. Genomic DNA was extracted from the sorted cells using the DNeasy Blood and Tissue Kit (catalog number 69504, Qiagen, Dusseldorf, Germany). PCR products were purified using the Universal DNA Purification Kit (TIANGEN, Beijing, China) following the instructions. The PCR products were then ligated to adapters, and sequencing was performed on the Illumina HiSeq X Ten platform.
2.4. Data Analysis for the PAM Library and Edited Window Library
In processing the PAM library data, we summarized the motifs based on the read-editing efficiency. The context plot was generated using the ggseqlogo package in the R software (version 4.2.3). To determine the editing efficiency, we extracted the reads with editing efficiency and calculated the proportion of these edited reads under the same barcode for both ends. We then classified them based on their PAM types. For the editing window-related library, we extracted the editing windows from the edited reads and performed the analysis. Similarly, for context plotting, we used the ggseqlogo package in the R software (version 4.2.3). Regarding editing efficiency, we determined it by calculating the proportion of edited reads to all reads under the same barcode.
2.5. Off-Target Detection by Whole Genome Sequencing (WGS)
We sorted individual K562 cells into 96-well plates, and subsequently, cell clones were cultured until confluence was achieved. These single clones of K562 cells were then allocated into three experimental groups. The transfection of the editor and sgRNA was performed using the Nucleofector®4D. WGS was executed with an average coverage of 50×, employing the BGI DNBSEQ-T7 platform.
2.6. fRYdCas9 Could Be Used in Mouse Embryo Editing
For fRYdCas9 mRNA, we linearized fRYdCas9 plasmids as templates, including a T7 promoter by a restriction enzyme for IVT processes. MESSAGE mMACHINE T7 ULTRA kit (Life Technologies, Waltham, MA, USA) was used for fRYdCas9 mRNA generation. For the IVT of sgRNA, The T7 promoter was added to the sgRNA template by the PCR amplification of px330 (a gift from Feng Zhang; Addgene plasmid #42230), using the primers listed below (
Table 1). The T7-sgRNA PCR product was purified and used as the template of IVT using the MEGA shortscript T7 kit (Life Technologies, Waltham, MA, USA). All IVT products of mRNA and SgRNA were purified using the MEGA clear kit (Life Technologies, Waltham, MA, USA) and eluted in RNase-free water.
During the injection, embryos were transferred into a droplet of M2 medium containing 5 μg/mL cytochalasin B (CB), and the FemtoJet microinjector (Eppendorf, Hamburg, Germany) was set to a constant flow. The injected embryos were cultured in KSOM medium with the amino acid at 37 °C under 5% CO2 for 2 h and then transferred into the oviducts of pseudo-pregnant ICR females at 0.5 dpc.
For embryo genotyping, in E4.5, a single blastocyst was transferred into 4 µL of medium and was crudely extracted, according to the manufacturer’s instructions, using the Crude DNA Extraction Kit (catalog number P072, Vazyme, Nanjing, China). The nested PCR of targeting loci was performed for embryo cell genotyping. In the first round of PCR amplification, Extaq (Takara, Shiga, Japan) was activated at 95 °C for 3 min, then PCR was performed for 30 cycles, at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, and a final extension was carried out at 72 °C for 5 mins after the cycles. The second round of PCR was performed using the same program with an inner nested primer. The PCR products were purified for Sanger sequence genotyping.
2.7. Statistical Analysis
For all statistical analyses in this study, we utilized R software (version 4.2.3). All tests were two-sided, and significance levels were denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
4. Discussion
By replacing the core components of fdCas9 with the PAM-less characteristics of RYdCas9 and the dimerization-mediated cleavage of FokI, we have engineered a highly precise editor, fRYdCas9, which significantly broadens the scope of targetable genomic loci while ensuring high fidelity. Although RYdCas9 has been successfully fused with FokI for gene editing in plants [
20], a more comprehensive understanding of its editing characteristics and potential off-target effects is still needed, especially when applied to human cells. In our research, the fRYdCas9 variant demonstrated access to more editing loci, showing a similar editing efficiency to fdCas9. Notably, the fRYdCas9 with a spacer length of 16-nt exhibited the highest editing efficiency. During library screening and WGS analysis, no PAM restriction, targeting motif preference, or minimized off-target effects were observed. The high-precision editor fRYdCas9 holds substantial potential for applications in biomedicine.
In our research, fRYdCas9 exhibited a nearly PAM-less editing characteristic, thereby avoiding the wide-ranging off-target effects of RYCas9. This suggests a more flexible PAM requirement for highly precise gene editing compared to other reported high-fidelity gene editors. The on-target outcomes observed for fRYdCas9 within the library show that FokI endonuclease exhibits no sequence preference for cleavage. Additionally, targeting outcomes from the PAM library demonstrate that fdCas9 can target sites with non-2 NGG PAM types. This observation aligns with existing literature highlighting Cas9’s proficiency in targeting atypical PAMs, such as NGA or NAG [
21,
22]. Importantly, these findings provide additional confirmation of the robustness of the meticulously constructed library.
In the field of genomic off-target detection, various methods exist, each with its distinct focus [
23]. Our study employed a conventional method for detecting off-target effects, involving the generation of monoclonal cell lines, gene editing by editors, and subsequent WGS. To mitigate external noise impact, we established the K562 monoclonal cell line through the isolation of a single-cell clone. Genomic sequencing was then conducted on the edited cells to identify potential off-target effects. In the fRYdCas9 group, we observed that the occurrence of off-target effects did not exhibit any correlation with the sgRNA sequences. This suggests that even in the presence of sgRNAs with a pronounced off-target tendency, fRYdCas9 can effectively constrain their off-target effects. Conversely, for RYCas9, although it has broadened its applicability, it has concurrently led to an increased incidence of off-target effects. We noted a greater number of off-target occurrences specifically associated with the PAM. Notable PAM sequences for off-target sites emerged, including motifs such as CAG, GAG, TAG, and CAA, among others. Our findings reveal that while RYCas9 effectively broadens its targeting scope, offering a beneficial complement for sites beyond the reach of certain Cas9 variants, it also introduces a heightened incidence of off-target effects. Our research indicates that the distinctive sgRNA usage rules of fRYdCas9 are capable of eliminating sgRNA-dependent off-target effects.
The high-fidelity characteristic of fRYdCas9 may be attributed to its dual-sgRNA binding requirements. This suggests that DNA cleavage by fRYdCas9 requires a 56bp match (comprising sgRNA length and spacer length) in the target region, which is significantly stricter than the binding requirements of wild-type Cas9 or RYCas9, which only require a 20nt match, thereby increasing its editing specificity. Nevertheless, despite its nearly PAM-less and high-fidelity editing features, fRYdCas9 still poses challenges in in vivo therapy delivery due to its large size. At present, it might be more suitable for genetic disease that could be handled at the cellular level, such as hematopoietic stem cell mediated-blood cancer therapy. Further procedures, such as split delivery methods, could be applied to enhance the delivery capability of this system and contribute to more precise gene editing in future applications.
In summary, the incorporation of dimerized FokI into the RYdCas9 variant results in the development of fRYdCas9, which exhibits several notable advantages compared to RYCas9. These advantages include improvements in the PAM feature, targeting motif preference, and reduction in genomic off-target effects. It would present an additional, safer alternative for forthcoming gene therapy or other applications, particularly in scenarios where edits from alternative high-fidelity Cas9 variants could be introduced. This advancement positions fRYdCas9 as a potentially high-fidelity editor in the context of therapeutic applications.