Responsive to synthetic NFAT CasRx

There is 1) a type of promoter with high specificity to a particular type of transcription factor, in this case, the synthetic NFAT, it achieves this by having various upstream elements of a small DNA sequence that contains the TATA box where polymerase II will bind 2) downstream, it contains a CasRx gene , a HA tag, an IRES2 RBS and a mCherry fused with NLS .

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1335
Illegal EcoRI site found at 1755
Illegal PstI site found at 4754
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1335
Illegal EcoRI site found at 1755
Illegal PstI site found at 4754
Illegal NotI site found at 141
Illegal NotI site found at 211
Illegal NotI site found at 506
Illegal NotI site found at 576
Illegal NotI site found at 3744
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1335
Illegal EcoRI site found at 1755
Illegal BamHI site found at 3735
Illegal BamHI site found at 3809
Illegal XhoI site found at 806
Illegal XhoI site found at 3210
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1335
Illegal EcoRI site found at 1755
Illegal PstI site found at 4754
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1335
Illegal EcoRI site found at 1755
Illegal PstI site found at 4754
Illegal NgoMIV site found at 1023
Illegal AgeI site found at 7
Illegal AgeI site found at 347
Illegal AgeI site found at 372
Illegal AgeI site found at 712
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

CasRx is a highly effective RNA-targeting molecule with nuclease activity specifically against single-stranded RNA, making it a versatile tool for gene silencing and antiviral therapies. One of its major advantages over RNA interference (RNAi) is its lower off-target activity, providing more precision in targeting specific RNA sequences. As a viral protein, CasRx operates independently of the host's cellular machinery, which reduces variability and enhances efficiency.

CasRx is the smallest member of the RNA-targeting CRISPR family, allowing for easier delivery into cells while maintaining high specificity and efficiency in cleaving target RNA. Its minimal off-target effects and high efficiency make it ideal for large-scale transcriptome screening, precise gene silencing, and targeting viral RNA in therapeutic applications.

A key advantage of CasRx over other CRISPR systems is its simplicity--it does not require a PAM sequence or a tracrRNA, making it one of the most straightforward CRISPR systems to implement. CasRx can target virtually any RNA sequence when paired with a guide RNA (gRNA) that is complementary to the target.

In the described system, an mCherry fluorescent protein is separately expressed and directed to the nucleus via a nuclear localization signal (NLS). The purpose of mCherry is to act as a reporter, enabling independent monitoring of the CasRx activity and target RNA levels. This separation of signals allows for precise tracking of the effector molecule (CasRx) and the concentration of the target RNA, enhancing the accuracy of the experiment.

By monitoring mCherry fluorescence in the nucleus, researchers can confirm the presence of CasRx without interference from the target RNA cleavage activity, ensuring clear and accurate results in gene silencing studies or antiviral applications.

Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell. 2018 Apr 19;173(3):665-676.e14. doi: 10.1016/j.cell.2018.02.033. Epub 2018 Mar 15. PMID: 29551272; PMCID: PMC5910255.

Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS. Methods for in vitro CRISPR/CasRx-Mediated RNA Editing. Front Cell Dev Biol. 2021 Jun 11;9:667879. doi: 10.3389/fcell.2021.667879. PMID: 34178991; PMCID: PMC8226256.

Engineering and structural experiment process

BUILDING CAS13D IN 10REPMIN FLUC

CasRx Insert Design

Three inserts were ordered from the supplier for the CasRx constructs: CasRx1: This insert contains the first half of the effector protein with a Kozak sequence for efficient mammalian expression. CasRx2: This insert includes the second half of the effector protein, fused with an HA tag at the C-terminal end. CasRx3: The third insert incorporates an IRES sequence and an mCherry reporter, also tagged with a nuclear localization signal (NLS) at the C-terminal end.

The inserts were designed to have overlapping sequences with each other as well as with the borders of the vector after the removal of the luciferase gene. The vector pNFAT-RE-Luc10, provided by professor Meško and her team, contains a minimal promoter that exhibits high specificity for a synthetic NFAT transcription factor and the luciferase gene downstream. Two restriction enzymes, PspXI (upstream) and FseI (downstream), were identified at the gene borders. Additionally, it was ensured that after cleavage, the vector would have overlapping sequences with CasRx1 and CasRx3 inserts. After initially failing to identify the correct construct during the testing phase, we reassessed our plasmid synthesis strategy. Fortunately, we received a plasmid from professor Balázsi and his team, which included both the mCherry reporter gene and the desired CasRx. However, their construct was controlled by a different regulatory mechanism than ours. Additionally, CasRx in their plasmid had a nuclear localization signal (NLS), while mCherry did not.

To isolate the mCherry and CasRx segments from their plasmid, we designed two primers, each with dual functionality. The forward primer contained a vector-specific sequence, the PspXI restriction site, and the NLS, while the reverse primer included an HA tag, a stop codon, and the FseI restriction site. These restriction sites were added to facilitate seamless cloning into our vector. Due to the differences in their design, we had to adjust our final construct: mCherry was placed at the front, followed by a P2A peptide, and then CasRx.

Since no inserts were successfully amplified during the initial building stage, we designed a new set of primers, called "half primers." These primers were shorter than the original ones, containing only half of the non-vector-specific portion. Our reasoning was that by using these half primers first, we could introduce part of the new sequence into the insert. In a second step, we would then add the full primers, giving them a better chance to anchor themselves to the template DNA and successfully amplify the entire sequence.

Build

Vector Preparation For the preparation of the vector, a double digestion was initially performed using PspXI and FseI on the pNFAT-RE-Luci10 construct, followed by gel extraction to isolate the backbone. Hifi For the construction of the final product, HiFi assembly was chosen. The three different inserts and the vector were mixed in a 2:1 molar ratio and incubated at 55°C for 15 minutes. After that, the ligation mixture was transformed into DH5α cell lines for amplification.

Long PCR We prepared the reaction by mixing the PCR Master Mix, the CasRx construct, Q5 High GC Enhancer, and the two primers. The initial PCR cycle consisted of three steps: denaturation at 98°C, annealing at 68°C, and extension at 72°C. This initial set of three cycles was designed to favor amplification of the vector-specific portion of the primers. Next, we performed an additional 17 cycles with denaturation at 98°C and a combined annealing and extension step at 72°C. This phase was optimized to amplify the full primer sequence, including the non-vector-specific regions, ensuring efficient amplification of the entire construct.

The reaction with half primers was similar to that with full primers. Initially, we introduced the half primers to the reaction, performed the first three cycles optimized for better specificity, and then carried out an additional 10 cycles. Afterward, we introduced the full-length primers and continued with another 17 cycles, similar to the previous reaction.

Testing the Construct

The pNFAT-RE-Luci10 vector was sequenced by the original researchers to confirm that no mutations were present in the overlapping regions. To differentiate suitable colonies from background colonies, alkaline lysis was performed on various colonies, which were then screened via restriction digest assays.

By performing restriction digest assays , if the Hifi assembly was succesful , cutting with the FseI and PspXI I, two pieces of 4445bp and 4841bp should result.

Finally, after screening numerous colonies, two of them exhibited the expected cutting pattern indicative of the desired construct. This remained consistent even after conducting multiple restriction digests with various enzymes on both colonies, confirming the presence of the original CasRx construct.

After performing Long PCR and running the sample on a gel, we did not observe the expected results. Despite a second attempt at performing PCR to insert the sequences, we were still unable to detect any amplified products.

Learning Outcomes:

Through the construction of this plasmid, we learned several important techniques and lessons. One of the initial challenges we faced was that following the instructions for the IDT suspension resulted in a sample with very low concentration, which was unsuitable for HiFi assembly, as denser samples are required. This experience taught us the importance of optimizing sample conditions before proceeding with assembly.

Additionally, we developed skills in screening colonies and distinguishing between successful and background colonies. We also gained hands-on experience in generating and applying a HiFi assembly protocol, significantly enhancing our understanding of molecular cloning strategies and their practical applications in genetic engineering.

"Probably the most important lesson learned was that, with enough effort, any plasmid can eventually be constructed. Lastly, we realized the importance of having backup plans and being ready to adjust strategies at the last minute if experimental conditions are not favorable