Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system

doi:10.21203/rs.3.rs-3590593/v1

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Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system

https://doi.org/10.21203/rs.3.rs-3590593/v1

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published 25 Jun, 2024

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The recently developed CRISPR activator (CRISPRa) system uses a CRISPR-Cas effector-based transcriptional activator to effectively control the expression of target genes without causing DNA damage. However, existing CRISPRa systems based on Cas9/Cas12a necessitate improvement in terms of efficacy and accuracy due to limitations associated with the CRISPR-Cas module itself. To overcome these limitations and effectively and accurately regulate gene expression, we developed an efficient CRISPRa system based on the small CRISPR-Cas effector Candidatus Woesearchaeota Cas12f (CWCas12f). By engineering the CRISPR-Cas module, linking activation domains, and using various combinations of linkers and nuclear localization signal sequences, the optimized eCWCas12f-VPR system enabled effective and target-specific regulation of gene expression compared with that using the existing CRISPRa system. The eCWCas12f-VPR system developed in this study has substantial potential for controlling the transcription of endogenous genes in living organisms and serves as a foundation for future gene therapy and biological research.

Biological sciences/Biotechnology/Gene therapy/Targeted gene repair

Biological sciences/Molecular biology/Epigenetics

The CRISPR system is a bacterial and archeal defense system that recognizes and cleaves invading viral DNA sequences using guide RNA-based endonuclease properties^{1, 2, 3}. Recently, the CRISPR activator (CRISPRa) and inhibitor (CRISPRi) systems have been developed based on their ability to recognize target DNA sequences through RNA reprogramming, allowing for the specific regulation of gene expression while avoiding DNA cleavage, which can cause serious errors within the living system^{4, 5, 6, 7}. The CRISPRa or CRISPRi system has evolved from the typical CRISPR-Cas9 system in that the endonuclease function has been eliminated and has been developed in a form in which the transactivator elements are tethered⁸. Effective regulation of gene expression can be achieved through the binding of CRISPR-conjugated transactivator elements in the optimal position upstream of the target gene's transcription start site (TSS)⁹. These molecular regulators have also been widely applied to various orthogonal modules of the Cas12 family for precise and effective gene expression control^{10, 11, 12, 13}. However, traditional CRISPR-Cas effectors have limitations in gene expression regulation, including weak activity or unsuitable sizes for adeno-associated virus (AAV) loading, depending on their specific module characteristics¹⁴. In this study, we addressed the weaknesses of the existing CRISPR activation system by introducing engineering modifications to both the guide RNA and Cas protein based on the recently discovered CRISPR-Cas12f effector for mammalian system application^{15, 16, 17, 18}. The CRISPR-Cas12f system possesses unique features compared to previously reported CRISPR-Cas systems, as it consists of very small domains and can recognize complementary target DNA in a homodimeric form¹⁹. Furthermore, the protospacer region, from the protospacer adjacent motif (PAM) recognition site to a distant point, is highly sensitive to mismatches between the guide RNA and target DNA, leading to exceptional target specificity²⁰. The gene expression activation system developed in this study, which was optimized for gene expression regulation using Cas12f from Candidatus Woesearchaeota archaeon (CWCas12f), was improved by introducing additional CRISPR component engineering. This system is based on the inactivated dCRISPR-Cas12f module, in which the viral component VP64, P65 and Rta (VPR) is directly tethered through a flexible linker (referred to as eCWCas12f-VPR). Notably, eCWCas12f-VPR demonstrated an average gene expression regulation efficiency several times higher than that of conventional CRISPR activator. Furthermore, it exhibited highly specific properties when targeting various genes in human-derived cell line. The improved eCWCas12f-VPR developed in this study demonstrates promising potential as a gene expression regulation tool for efficient and specific applications in biological systems. Additionally, the eCWCas12f-VPR system, based on the preserved functionality of the recognition and nuclease domains of RNA-based endonucleases, can provide valuable insights into the diverse applications of the newly discovered gene-editing tools such as Cas12^{21, 22, 23, 24}, TnpB^{25, 26}, and Fanzor²⁷, which are currently gaining prominence in biomedical applications.

Optimization of CRISPR-Cas12f-based gene activation system

To construct an effective CRISPR activator to induce the expression of target genes inside cells, we connected the trans-activator domain VPR to the dead version of the CWCas12f (dCWCas12f, D354A) system¹⁵. The CRISPR-Cas12f system, in contrast to previously reported Cas12 effectors, has a relatively small size, which is pertinent for the core function of target DNA binding and cleavage within a single component¹⁹. Ideally, it recognizes the target DNA in a homodimeric form, allowing the recruitment of two copies of the VPR module to the upstream region of the target gene per Cas12f molecule (Fig. 1a). To optimize the operation of the dCWCas12f-VPR system within target DNA sequences, various configurations of linkers and nuclear localization signals (NLS) were employed to connect dCWCas12f and VPR (Fig. 1b; Supplementary Fig. S1). Additionally, plasmids inducing fluorescent signals within human-derived cells were co-delivered to compare their activities (Fig. 1c, d). When analyzing the activity of the dCWCas12f-VPR system in human-derived HEK293FT cells following transfection with plasmids harboring specific target sites (upstream sequence for TSS of NLRC4 and KLH29 genes) (Supplementary Table S1), normalizing the activity to GFP fluorescence intensity and comparing it to dTomato fluorescence intensity, it was observed that V3 exhibited relatively higher values (Mean fluorescence intensity 1.82 (24 h)–2.59 (48 h) for NLCR4, 0.70 (24 h)–1.60 (48 h) for KLH29), as shown in Supplementary Fig. S1. When different guide RNA versions (ge4.0 or ge4.1) were used, the ge4.1 version of the guide RNA yielded a relatively higher average fluorescence intensity (Fig. 1c, d). Therefore, we conducted additional follow-up experiments using the ge4.1 version of the guide RNA and the V3 version of the dCWCas12f-VPR system, which exhibited the highest value of average dTomato fluorescence for the various genes analyzed.

Further engineering of CRISPR-Cas12f system for enhanced regulation of gene expression

In the previous experiment, the optimized combination of linker types and NLS with the V3 version of the dCWCas12f-VPR system, along with the ge4.1 guide RNA, was used to improve the efficiency of gene expression activation on actual genomic sites in human-derived cells (Fig. 1e, f). The introduction of amino acid mutations in a similar Cas12f system led to an increase in the targeting efficiency of Cas12f toward the target DNA sequences¹⁸; therefore, identical mutations ((D171R, T175R, E179A) or (D171R, T175R, K358R, E556R)) were introduced at conserved amino acid positions within the dCWCas12f-VPR system to further enhance its efficiency (Supplementary Fig. S2a). Moreover, these mutant versions were further combined with the FUS-IDR domain²⁸, which is known to recruit transcription initiation factors through phase separation, yielding four different versions (3.1–3.4) of the enhanced dCWCas12f-VPR system (Supplementary Fig. S2b). Using these four different versions, which were created via the introduction of various forms of mutations and FUS-IDR domain conjugation, gene expression activity in the upstream region of SOX10 in a human-derived cell line was compared and analyzed through tiling assays (Fig. 1e; Supplementary Fig. S3, Supplementary Table S1). When the 3.3 version of the dCWCas12f-VPR system was introduced into cells to induce gene expression, it consistently exhibited the highest average induction efficiency at various positions in the TSS upstream region (Fig. 1f). Additionally, this version exhibited the highest expression efficiency in the target site (sg3) of the TSS upstream region among the different versions. Notably, the enhanced 3.3 version of the dCWCas12f-VPR system (referred to as eCas12f-VPR) demonstrated an efficiency comparable to that of previously reported gene expression activation (CRISPRa) systems based on Cas9 or Cas12a (Fig. 1f).

Targeted activation of endogenous gene expression across multiple genes using Cas12f-based activator

We investigated whether the previously improved eCas12f-VPR system could universally induce gene expression at various genomic sites (CD2, CXCR4, HBB, IL1RN, ASCL1, and HBG) in human-derived cells (Fig. 2, Supplementary Table S1). To directly compare the regulatory effects of the CRISPR-Cas module on gene expression with existing CRISPR activators, we compared it with the well-characterized Cas12a system¹². The previously reported LbCas12a-VPR system recognizes a T-rich PAM (TTTV) similar to the PAM (TTTR) of the eCas12f-VPR system, facilitating the direct comparison of gene expression efficiency at the same genomic sites (Supplementary Fig. S4, Supplementary Table S1). Across all tested gene loci, the eCas12f-VPR system exhibited an overall superior gene expression amplification efficiency compared to the LbCas12a-VPR system. Application of the LbCas12a-VPR and eCas12f-VPR systems resulted in an average of 29.72-fold and 965.46-fold induction of gene expression, respectively. When applying the eCas12f-VPR system, the gene expression amplification for CD2, CXCR4, HBB, IL1RN, ASCL1, and HBG increased by an average of approximately 29.61-, 1.46-, 5.08-, 1.99-, 3.95-, and 242.90-fold compared to that of the LbCas12a-VPR system (Fig. 2a-f, Supplementary Table S2). Notably, for the target genes HBB, ASCL1, and HBG (Fig. 2c, e, f), for which the existing LbCas12a-VPR system was unable to induce significant gene amplification, the eCas12f-VPR system was effective in inducing gene expression. Similar to previous reports, for both the LbCas12a-VPR and eCas12f-VPR systems, we observed significant variability in the gene expression efficiency for different target DNA sequences within the TSS upstream region of each gene. Consequently, when comparing gene expression induction for the same target DNA sequences within the same gene (Supplementary Fig. S4), the eCas12f-VPR system demonstrated more effective gene expression than the LbCas12a-VPR system. Both systems exhibited significant efficiency variations depending on the target DNA sequence.

Highly specific activation of gene expression using Cas12f-based activator

To confirm both the effective gene expression amplification characteristics and specificity for the target genes, we compared the on/off-target gene expression profiles (on/off-target mRNA fold enrichment ratio) of the eCas12f-VPR system to those of the LbCas12a-VPR system (Fig. 3). We searched for common targets and respective off-target candidate sequences based on the PAM nucleotide sequences for various gene loci within human-derived cells (HBB, ASCL1, HBG, CD2) (Fig. 3a, e, i, m; Supplementary Table S3). Subsequently, we verified the induction of gene expression in both on- and off-target sequences. When the LbCas12a-VPR system was used, we observed average on/off-target ratios of 5.42-fold for HBB, 5.49-fold for ASCL1, 135.00-fold for HBG, and 298.88-fold for CD2 (Fig. 3b, f, j, n). However, when the eCas12f-VPR system was applied, the average on/off-target ratios were 23.18-fold for HBB, 61.60-fold for ASCL1, 11257.15-fold for HBG, and 250.71-fold for CD2 (Fig. 3c, g, k, o). When using the eCas12f-VPR system to induce gene expression, we observed an overall increase in target specificity based on the on/off-target ratios, with an average improvement of 26.06-fold when compared to the LbCas12a-VPR system.

The development of CRISPR activator/inhibitor systems that enable the control of gene expression in target genes without altering the underlying DNA sequence, as opposed to traditional CRISPR endonuclease forms, has immense potential for future therapeutic and biosystem applications. As part of these efforts, the eCas12f-VPR system developed in this study, based on the engineered CWCas12f module, demonstrated superior target gene specificity and efficacy for gene expression activation compared to those of the existing CRISPR-Cas modules. In particular, when directly compared to the LbCas12a-VPR system, which recognizes T-rich PAM sequences (TTTR), eCas12f-VPR exhibited increased efficiency (average increase of 32.48-fold) and specificity (average increase of 26.06-fold) for various genes in human-derived cell lines. These results suggest that the eCas12f-VPR system can be effectively and precisely applied for gene expression regulation in various cellular systems, offering the potential for a wide range of applications, such as screening and gene therapeutics. However, the TSS region of the target gene should be clearly defined before cell treatment for the effective application of the eCas12f-VPR system to cellular targets. This will enable the selection of effective target DNA sequences in the TSS upstream region for better control of gene expression.

In this study, the VPR domain was used as a transactivator connected to the CRISPR-Cas12f system to induce specific gene expression; however, other domains, such as KRAB^{29, 30}, can be used to achieve effective and specific gene expression inhibition in further studies. Additionally, recent examples, including the use of a combination of KRAB-DNMT3A and VPR-TET1 based on dCas9 modules³¹, suggest that linking both transcriptional and epigenetic modification domains based on the dCas12f effector can effectively and sustainably regulate specific gene expression in cellular targets. The eCas12f-VPR system used in this study is highly compact, allowing the addition of such factors in an all-in-one format that can be incorporated into effective delivery vehicles, including AAV systems¹⁴. This can potentially enable highly efficient control of gene expression through effective delivery in biological systems. In addition, beyond the eCas12f-VPR system, the use of newly discovered forms of other RNA-guided effectors, such as OMEGA^{25, 26, 27}, based on the context of trans activation system developed in this study, can be expected to enhance the efficiency and specificity of gene expression regulation. Conclusively, the eCas12f-VPR system developed in this study provides a foundation for future applications in various biomedical fields, including molecular level-based screening, identification of gene functions, and disease modeling. Furthermore, this targeted and specific gene expression control technology will likely lay the groundwork for the development of gene therapies applicable to humans.

Design and cloning of various transcriptional activator and guide RNA expression vectors

Cytomegalovirus (CMV) promoter-based expression vectors for dCWCas12f(D354A)-VPR, dLbCas12a(D832A)-VPR, and dSpCas9m4(D10A/D839A/H840A/N863A)-VPR were used to facilitate targeted gene expression in human-derived cell line (HEK293FT, Invitrogen (R70007)). LbCas12a(D832A)-VPR (Addgene no. 104567) and SpCas9m4(D10A/D839A/H840A/N863A)-VPR (Addgene no. 63798) were constructed and used for experimental purposes. To optimize the efficiency of dCWCas12f-VPR, different versions of dCWCas12f-VPR with varying linker lengths and additional NLS were designed (Fig. 1b). eCWCas12f (D171R/T175R/E179A)-VPR was cloned from dCWCas12f(D354A)-VPR v3 (Supplementary Fig. S2). For targeted gene activation, targets at various positions were selected (Supplementary Table S1), and guide RNAs (gRNAs) were carefully designed to corresponding these positions. These targets were strategically positioned at various distances from the TSS. The target gene expression levels were detected using optimally designed qPCR primers (Supplementary Table S2). As comparative controls, gRNAs for dSpCas9m4-VPR, crRNAs for dLbCas12a-VPR and dCWCas12f-VPR were constructed. The gRNA expression vector, driven by the U6 promoter, was designed and manufactured to allow for the facile replacement of the protospacer with restriction enzymes, ensuring adaptability to various target nucleotide sequences.

Cell culture and transfection

The human-derived cell line HEK293FT was purchased from Invitrogen (R70007) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (both sourced from Gibco) at 37°C in the presence of 5% CO₂. The cells were subcultured every 48 h to maintain a 70% confluency. For targeted gene activation, the cells were transfected into 24-well plates, with each well initially seeded with 2 × 10⁵ cells. At 24 hours after seeding, each gene targeting gRNA expression plasmids (240 pmol), transcriptional activator expression plasmids [dSpCas9m4-VPR (Addgene no. 63798), dLbCas12a-VPR (Addgene no. 104567), and various dCWCas12f-VPR (developed in this study)] (60 pmol), and reporter plasmids (40.7 fmol) were delivered to the cells at 60% confluency with 1.5 µl lipofectamine 3000 reagent and 1 µl p3000 reagent (Thermo Fisher Scientific), according to the manufacturer’s protocols. RNA extraction and fluorescence assays were performed 24–48 h after transfection.

Fluorescence reporter assay

Reporter assays were performed by targeting various versions of dCas12f-VPR (v1, v2, v3, v4, v5, and v6) to a mimi-CMV-based dTomato expression reporter system, driving the expression of a fluorescent reporter. A dTomato expression reporter systems, which harbor appropriate target sequence, were constructed based on Addgene plasmid (#47320) to test the transcriptional activation of the dCWCas12f-VPR. The binding sequences of dCWCas12f-VPR are listed in Supplementary Table S1. Transfected cells were observed using a fluorescence microscope 24–48 hours after transfection. The average fluorescence intensity was quantified by calculation based on measurements by using ImageJ software for randomly selected fluorescence image areas.

RNA extraction and quantitative polymerase chain reaction for evaluating transcriptional activation

Total RNA was extracted from cultured cells 48 h post-transfection using the RNeasy Mini Kit (Qiagen). Subsequently, 1 µg of RNA from each sample was used for cDNA synthesis using the iScript cDNA synthesis Kit (BioRad), according to the manufacturer’s protocol. The resulting cDNA was employed in each quantitative polymerase chain reaction (qPCR) cycle. The quantification of specific gene expression was achieved through qPCR using the THUNDERBIRDTM Next SYBR® qPCR Mix (TOYOBO). Subsequently, qPCR was performed and analyzed using a CFX96 Real-Time PCR Detection System (BIORAD). To evaluate transcriptional activation, cDNA was amplified using the qPCR primers listed in Supplementary Table S2. The expression levels of each targeted gene were normalized to the cycle threshold (Ct) of the housekeeping gene GAPDH, yielding ΔCt values. ΔCt was calculated as ΔCt = Ct (target gene) – Ct (housekeeping gene (GAPDH)), and ΔΔCt was determined as ΔΔCt = ΔCt (experimental sample) – ΔCt (control sample), with the non-targeting experimental sample used as the control. All reactions were performed in triplicate, and the relative expression of each sample was computed as 2^(-ΔCt) or 2^(-ΔΔCt).

Off-target analysis

Potential off-target site candidates were selected using Cas-OFFinder³² to identify genome-wide off-target sites (Supplementary Table 3). Each off-target candidate was selected as a high probability candidate and is located near from a coding gene. To validate off-target gene activation induced by CRISPR activators, qPCR was conducted for each on-target and predicted off-target candidate sites using the synthesized cDNA from extracted total RNA.

Acknowledgements

This research was supported by grants from the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare). (22A0203L1). This study was also supported by the Chung-Ang University Research Scholarship Grants in 2023.

Author contributions

Conceptualization, Y.O., K.P.K. and S.H.L. ;Methodology, Y.O., K.P.K. and S.H.L. ;Software, Y.O. and S.H.L. ;Validation, Y.O. and S.H.L. ;Formal Analysis, Y.O. and S.H.L. ;Investigation, Y.O. and S.H.L. ;Resources, Y.O., K.P.K. and S.H.L. ;Data Curation, Y.O., K.P.K. and S.H.L. ;Writing-Original Draft, Y.O. and S.H.L. ;Writing-Review & Editing, Y.O., K.P.K. and S.H.L. ;Visualization, Y.O. and S.H.L. ;Supervision, Y.O., K.P.K. and S.H.L. ;Project Administration, Y.O., K.P.K. and S.H.L. ;Funding Acquisition, K.P.K. and S.H.L.

Data availability

All relevant data supporting the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

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You are reading this latest preprint version

Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Optimization of CRISPR-Cas12f-based gene activation system

Further engineering of CRISPR-Cas12f system for enhanced regulation of gene expression

Targeted activation of endogenous gene expression across multiple genes using Cas12f-based activator

Highly specific activation of gene expression using Cas12f-based activator

Discussion

Methods

Design and cloning of various transcriptional activator and guide RNA expression vectors

Cell culture and transfection

Fluorescence reporter assay

RNA extraction and quantitative polymerase chain reaction for evaluating transcriptional activation

Off-target analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1