Development of Alternative RTs

Introduction

Prime editing is a versatile innovation that enables a wide array of gene modifications, including base-to-base conversions, insertions, and deletions, all without the need for double-stranded breaks or donor DNA templates. It relies on specialized prime editors (PEs), which is a fusion of reverse transcriptase (RT) enzymes with nickase Cas9 (nCas9), and the prime editing guide RNA (pegRNA). This RNA encodes both the target sequence and the new sequence required to introduced desired mutations, offering remarkable target specificity and diverse real-world applications.

Types of Prime Editors

The pioneer of prime editing (known as Prime Editor 1 or PE1) comprises MMLV RT fused to nCas9 (H840A) [1]. While it has shown limited efficacy in facilitating precise insertions and deletions, it prompted interest in refining the RT component to enhance DNA synthesis. As such, numerous studies have documented mutations that inactivate RNAse H activity [2], increase thermostability [3], and enhance DNA-RNA substrate affinity [4] and processivity [3]. These discoveries culminated in the development of the pentamutant MMLV RT (D200N/L603W/T330P/T306K/W313F) incorporated into PE1, which is now recognized as Prime Editor 2 (PE2). Unlike PE1, PE2 exhibited notable enhancement, with improvements ranging from 1.6 to 5-fold when executing point mutations, targeted insertions, or deletions [5].

Subsequently, PE3 and PE3b incorporate an additional single guide (sgRNA) that cleaves the unedited strand at a location distant from the editing site. PE3b's sgRNAs are designed with spacers tailored to the edited strand, but not the original allele. This strategic design prevents sgRNA cleavage until the PAM strand has been edited due to mismatches between the spacer and the unedited allele. When compared to the PE3 system, PE3b demonstrated a notable reduction in the average number of insertions and deletions, without any discernible decrease in editing efficiency [6].

Existing Research on Reverse Transcriptase

The PE has been the focus of extensive research, with scientists modifying its structure through truncation, addition of sequences, and the integration of multiple pegRNAs. Despite these efforts, MMLV RT remains the most utilised RT for prime editing. While many studies aim at enhancing various characteristics of the RT, several limitations persist that could potentially hinder the activity and precision of prime editing. The overall length of the reverse transcriptase template (RTT) and the presence of a flap base before a guanine nucleotide (G) directly impact the accuracy of prime editing. Specifically, a longer RTT obstructs the read-through of the reverse transcriptase into the scaffold, whereas the presence of G before the edited flap leads to reduced prime editing efficiency [7].

Furthermore, research indicates that MMLV RT excels in implementing single base replacements compared to insertions and deletions, displaying a preference for A-to-G conversions. Notably, there are also challenges associated with achieving high prime editing efficiency at specific target sites, such as EMX1 and RUNX1. Additionally, the PE exhibits lower activity during the precise insertion of longer edits, such as a Flag epitope tag (24 base pairs, with an 18% efficiency), as well as in the case of an extended Cre recombinase loxP site (44 base pairs, with a 23% efficiency) [6].

Various research groups have explored diverse RT variants sourced from viruses and bacteria, including human foamy virus (HFV), human endogenous retroviral (HERV), and xenotropic murine leukemia virus-related virus (XMRV) RTs [8, 9]. The Marathon RT, derived from Eubacterium rectale, has also undergone frequent testing and optimization for in vitro laboratory applications [10].

While studies indicate significant efforts to identify and enhance alternative RTs, it is noteworthy that MMLV RT remains the most efficient choice for prime editing. However, it is important to acknowledge that MMLV RT does have its limitations, which can impact prime editing performance. To address this, we are conducting a screening of viral RTs to identify a new RT as a potential alternative to MMLV RT.

In our project, we evaluated eight viral RTs by comparing their editing efficiency against wild-type PE2 and PE2 with a truncated RNAse H region (PE-497). Previous research has shown that the deletion of the RNAse H region does not compromise prime editing activity in mammalian cells, which informed our decision to include this modification in our work [28].

Results and Discussion

To assess the performance of various RTs, we co-transfected them with pegRNA and ngRNA targeting the RNF2, HEK3, and EMX1 genomic loci in HEK293T cells. We estimated the editing efficiency using Sanger sequencing. As anticipated, truncation of the RNAse H region did not compromise prime editing activity and, in fact, slightly improved it at RNF2 and EMX1. Among the eight alternative RTs, PFV demonstrated the highest level of performance, achieving 28% (Figure 1), 52.5% (Figure 2), and 8% (Figure 3) on the RNF2, HEK3, and EMX1 genomic loci, respectively. Another noteworthy performer was MMTV RT, which exhibited a significant editing efficiency of 67% on HEK3, and 7% and 4% on RNF2 and EMX1, respectively.

Figure 1.Screening of RT variants for prime editing activity at RNF2 target site. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates;

Figure 2.Screening of RT variants for prime editing activity at HEK3 target site. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates.

Figure 3.Screening of RT variants for prime editing activity at EMX1 target site.

To further assess the performance of alternative RTs, we conducted in vitro prime editing using a TLR reporter plasmid, an advanced TLR system designed in our laboratory. Cell analysis was conducted at both 24 hours and 72 hours after transfection, with 24 hours providing a more stringent selection criterion. Figures 4 and 5 illustrate that PFV and MMTV outperformed other RTs in correlation with previous results.

Figure 4. Prime editing efficiency on TLR reporter plasmid 24hr after transfection. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates.

Figure 5. Prime editing efficiency on TLR reporter plasmid 72hr after transfection. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates.

The consistent and robust performance of PFV RT across three distinct endogenous sites and in the TLR reporter system positions it as an excellent candidate for serving as an alternative RT in prime editing.

In a study involving the Split-PE system, various RTs sourced from both bacterial and viral origins were screened, with HFV RT (1827 bp) being one of the options considered [8]. As depicted in Figure 6 below, HFV RT exhibited notably lower (or even negligible) editing frequency on RNF2, RUNX1, and HEK sites, respectively.

Figure 6. (c) PPE frequencies of seven non-MMLV RTs tested : Human codon-optimized non-MMLV RTs tested were from human foamy virus (HFV), human endogenous retrovirus K (HERV-K_con), lactococcal group II intron Ll.ltrB (LtrA), Thermosynechococcus elongatus group II intron (TeI4c), Methanosarcina aromaticovorans intron 5 (Ma-Int5), Geobacillus stearothermophilus GsI-IIC intron (GsI-IIC) and E. rectale (Eu.re.I2) group II intron (Marathon). n = 3, technical replicates. (d) Schematic showing the lengths of all non-MMLV RTs tested in (c) in comparison to MMLV-RT (without counting start codons) [8].

To assess the similarity between the HFV RT examined in the study and the PFV RT from our lab, we conducted a protein sequence alignment using Serial Cloner Software. The alignment results, illustrated in Figure 7, reveal a 97.44% similarity between the two sequences, with the main disparity being a truncation of 120 amino acids at the front end of the HFV. This incomplete sequence of HFV likely contributes to its lower prime editing efficiency.

Figure 7. PFV and HFV RT protein sequence alignment. Sequence one represents PFV while sequence two represents HFV.

In an attempt to replicate the study's results, we deleted 96 amino acids from the front end of PFV. As depicted in Figures 8 and 9, this led to a significant drop in efficiency, from 32.5% and 35.5% of the original full-length PFV RT to 13.5% and 5.5% with the 1-96 deletion on RNF2 and HEK3 target sites, respectively. This suggests that the deleted region plays a crucial role in the function of the RT.

Given our understanding that the RT structure contains an RNase H domain, we hypothesized that the removal of the RNase H domain in PFV will not significantly affect the gene editing efficiencies. RNase H is only for RNA template degradation and this domain is also inactive in the mutant MMLV RT of PE2. We opted to take a similar approach with the truncated MMLV RT and removed the RNase H domain of PFV (deletion 583-748). The results, as seen in Figures 8 and 9, indicated a comparable to full-length PFV level of editing efficiency.

Figure 8. Prime editing efficiency at RNF2. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates.

Figure 9. Prime editing efficiency at HEK3. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates.

Finally, to validate the performance of the newly identified PFV RT, we conducted a test at a therapeutic site, specifically targeting the single nucleotide polymorphism (SNP) responsible for Primary Open-Angle Glaucoma (POAG). This site was recommended for testing by a clinician after it was discovered by Gharahkhani, et al. [11], and pegRNA and ngRNA for prime editing were custom-designed in our laboratory. Figure 10 demonstrates the presence of editing at the target site, albeit at a lower efficiency.

Figure 10. Prime editing efficiency at HEK3. Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates.

Conclusion

In conclusion, we assessed eight RTs and identified PFV RT as a promising candidate, displaying noteworthy editing efficiency across three genomic loci, the TLR reporter system, and a therapeutic site. PFV RT can be developed similarly to MMLV RT by incorporating mutations to enhance processivity, structural stability, fidelity, or other pertinent characteristics relevant to prime editing. We anticipate that our findings will provide researchers with an alternative option and contribute to the continued improvement of PFV RT as has been seen with MMLV.

Prime editing in stem cells

Genome editing has a wide range of usage in biological research. This includes, but is not limited to, research from human disease, high throughput screening of identification and validation of novel therapeutic targets. Genome editing is usually carried out by gene mutations such as gene replacement and insertions frameshift.

Various techniques for genome editing in stem cells, such as ZFN (Zinc finger nuclease), TALEN (Transcriptase activator-like effector nucleases), CRISPR, and base editing, have yielded remarkable outcomes in the past [12]. Depending on the underlying working principles, these technologies are categorized into traditional genome editing and programmable nucleases.

ZFNs were the first programmable nucleases introduced for genome editing in stem cells. ZFN comprises of two domains, the DNA binding zinc finger domain and a nuclease domain derived from the FOKI restriction enzyme. Since 2009, ZFN has been utilized as Clinicals trials in USA and China for patients with Human immunodeficiency virus (HIV) infection, sickle cell anemia and transfusion dependent beta-thalassemia [12, 13]. Unfortunately, limitations lie within the design of the zinc finger domain.

TALENs were also first used for genome editing after the identification of DNA-recognizing bacterial proteins. TALENs, similar to ZFNs, contain a FOKI nuclease domain at their carboxyl terminal. In principle, TALENs can edit any DNA sequence while the drawbacks are as follows: large size of 6kb per pair which makes delivery difficult although achievable through codon divergent repeat variable di-residues (RVDs), adenoviral vectors or mRNA or protein-based gene transfer method. The current application of TELENs in stem cells has been shown in 3 phase I interventional clinicals trials for acute myeloid leukemia [12, 14].

CRISPR/Cas was developed with the discovery of an interrupted short tandem repeat sequence of nucleotide in Escherichia coli. The well-known CRISPR systems currently in use are divided into two major classes and encompass six types depending on their related Cas genes and organization of the corresponding loci. The CRISPSR/Cas9 system consists of two components, the Cas9 nuclease and a single guide RNA. However, CRIPR tools may cause severe adverse effects so there needs to be an implementation of anti CRISPR proteins, inhibiting the CRISPR system after [12, 14].

CRISPR Cas9 base editing causes irreversible converts to nucleotides in the targeted window without causing double strand breaks (DSBs). There are two base editors that have been reported thus far: including the cytidine base editors (CBEs) and adenosine base editors (ABEs). Base editing mobilizes the use of a dead form of Cas9 creating nicks on the opposite strand, manipulating the DNA repair machinery increasing the efficiency of editing and yielding of base editing. Hence, base editing technologies may be used to treat diseases caused by single nucleotide polymorphism (SNP), by correcting the mutations. Base editing has been carried out widely in stem cells. An example of application would be modifying induced Pluripotent stem cells (iPSCs) by delivering CBE and ABE with a 4D-nucleofactor system furthermore, base editing was also carried out in mouse embryos and there was generation of mutant disease in rabbits and pig’s model [12, 15].

The advantages of CRISPR over ZFN and TALEN was that CRISPR had a much simpler target design as compared to ZFN or TALEN as the target specificity relies on ribonucleotide complex and not protein or DNA recognition. The guide RNA can be designed easily and cheaply to target any sequence in the genome. Furthermore, CRISPR is very efficient as modifications can be directly injection of RNAs encoding CAS protein and guide RNA into the model’s embryo, by-passing the tedious transfection and selection process [16, 17].

However, with all the benefits there are still drawbacks. Off-site effects are the greatest hindrance in genome editing as mutations can be introduced into non-specific loci with similar but non-identical homology to the target sites [13, 17, 18]. Furthermore, CRISPR breaks both strand of DNA and carry out either non-homologous end joining, or homology directed repair which can either result in gene knockout due to indel mutation or require a DNA donor template and results in gene knock in. Both causes irreversible roles and life changing or threatening outcomes.

Thereby, the prime editor (PE) was introduced, the most recent addition of methods to genome editing and was developed based on CRISPR/Cas9. The PE consists of Cas9 fused with an engineered reverse transcriptase (RT) and a specialized guide RNA, prime editing guide RNA (pegRNA). Prime editing does not cause DSBs or require a donor DNA template as the pegRNA replaces the sequence of the target strand. Prime editing has been successfully shown in human hematopoietic stem cells [19] and human embryonic stem cells [20]. Therefore, the current challenge of prime editing in stem cells is the low editing efficiency. Many experimental analyses of prime editing efficiencies have shown that the editing efficiency is typically below 20% for immortalized cell lines and greatly various between target loci and cell types.

Our project thus aims to provide alternative so in probable future, improving the efficiency of Prime editing.

Two stem cell lines were tested, H9 and ATCC-ACS1030. H9 being a standard human embryonic stem cell and ATCC-ACS1030 is a human induced pluripotent stem cell (iPSC). These two cell types were selected due to pluripotency, the ability to indefinite self-renewal and the potential to differentiate into nearly all cell types [21]. Hence, the mutations of patient-derived iPSCs can be corrected and differentiated towards specific cell types for therapeutic purposes. Both cell lines were grown in mTeSR™ media and initially seeded with Rho kinase inhibitor 1 (Rock 1). Plates were coated with Matrigel to aid the adherence of stem cells.

All stem cells were transfected to introduce the plasmids required for prime editing. We tested various ways of transfection to enter the cell, namely by chemical transfection using Lipofectamine 3000 and electroporation using the Amaxa 4D nucleofector.

Lipofectamine is used for chemical transfection. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, entrapping the transfection payload (Figure 1b, c). By forming the lipid-nucleic acid complex, the cells will engulf the lipoplexes forming vesicles called endosomes, then travel into the cytoplasm (Figure 1d). Once in the cytoplasm, the lipoplex will start to break down, releasing nucleic acid into the cellular environment.

leading to the expression of the gene (Figure 1e). The cells were transfected with Lipofectamine 3000 in Optimem Media.

Figure 1. Pictorial visualization of mechanism of Lipofectamine [22].

Nucleofection is a transfection method that uses an electrical field to introduce nucleic acid into a cell. Similarly, there would be a formation of nucleic acid cell complex when the nucleic acid is mixed with prepared cells. There is also a special transfection enhancer or solution that is added to the mixture which stabilizes the nucleic acid and the interaction with the cell membrane. An electrical pulse will then be applied to the mixture, creating transient pores to allow the nucleic acid to enter the cells. Parameters such as the voltage, pulse and resistance will have to be optimized for different cell types and nucleic acids. Cell recovery will be crucial at this point, so the recovery medium is added to help cells return to normal membrane integrity. Subsequently, the gene will be expressed. The kit used was Lonza 4D-Amaxa nucleofector.

The optimization of prime editing in stem cells uses 3 plasmids: PE2, ngRNA and HEK3 pegRNA. The targeted site is HEK3. The plasmid containing ngRNA was incorporated with mCherry such that red fluorescence would be produced during successful transfection.

Comparing Figure 2 and Figure 3, the results for Lipofectamine 3000 were promising, which we then decided to further optimise it.

Figure 2. Results of Nucleofector.

Figure 3. Results of Lipofectamine 3000.

As for Amaxa Nucleofector, after transfection, there was no indication of a positive result as there was no red fluorescence. Thus, the Amaxa Nucleofector will not be used for the testing of RT variants was not optimised.

Lipofectamine 3000 has 2 reagents, the Lipo3000 and P300, the ratio of the 2 reagents was optimised by transfecting the cells. After 72 hours, they were sent for FACS and data was recorded to obtain the transfection efficiency. It was then sent for Sanger sequencing to obtain the editing efficiency.

Figure 4. Transfection efficiency%, for optimization P300 to Lipo3000 ratio of lipofectamine 3000 Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates

Figure 5. Editing efficiency%, for optimization P300 to Lipo3000 ratio of lipofectamine 3000 Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates

Based on the result from Figure 1, 2, 3, 4 and 5, there is a clear indication that the transfection efficiency correlates with the editing efficiency. The higher the rate of transfection, the higher the rate of editing. Furthermore, it can also be deduced that the optimal P300 to Lipo300 for HEK293T, H9 and ACS1030 are as follows: 1.5 to 1.5, 1 to 1.125 and 1 to 1.5 respectively.

Comparing Figure 6 and Figure 7, the transfection efficiency was higher when the media was changed at 12 hours after transfection. While the editing efficiency was higher when the media was changed 12 hours after transfection, there may be underlying factors that affect its editing efficiency. Therefore, as per the previous conclusion that transfection efficiency was proportional to editing efficiency, the media was changed for further experiments at 24 hours after transfection instead.

Figure 6. Transfection Efficiency%, optimization of hours before changing media after transfection Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates

Figure 7. Editing efficiency%, optimization of hours before changing media after transfection Bar graphs show the mean value and error bars indicate S.D. of n = 3, independent biological replicates

After transfection using Lipofectamine 3000 was optimized, the other RTs were put to test. Results are as follows:

Figure 8. Editing efficiency% of 3 RT (PE2, PE497, PE6d)

Based on the editing efficiencies of the different RTs of PE2, PE 497, and PE6d, the highest editing efficiency is different for each cell line, PE497 for HEK293T, PE6d for H9 and PE2 for ACS1030.

Further on, there will be testing on different RT from different sources. The list are as follows: Respiratory syncytial virus (RSV) (BBa_K4830033), truncated duck Hepatitis B virus RT (miniRT2) (BBa_K4830030), Prototype foamy virus (PFV) (BBa_K4830015), Cauliflower mosaic virus (CaMV) (BBa_K4830032), Bovine leukemia virus (BLV) (BBa_K4830031), Li.ltrA (BBa_K4830017), Mouse mammary tumor virus (MMTV) (BBa_K4830018) and Line 1 (BBa_K4830034). This is to explore alternate options apart from PE (MMLV RT).

Different sites would also be tested for the following: Primary Open-Angle Glaucoma (POAG) and RING (really interesting new gene) finger (RNF).

Further findings will be reported during Grand Jamboree.

Prime editing in yeast & bacteria

BACTERIA

Introduction

Gene editing in bacteria is not new, and numerous attempts have been made to alter the bacterial genome for industrial applications such as biofuel production [23] and the production of valuable chemicals [24]. Unfortunately, the development of bacteria into major cell factories for bio-production is greatly hindered by low editing efficiencies and the need for a donor DNA template [25]. Due to tightly interconnected metabolic pathways, accurate regulation of these reactions in bacteria to achieve such purposes requires efficient, precise, and simple gene editing tools.

Since the discovery of Cas nucleases, CRISPR-Cas systems are used to edit the bacterial genome by introducing double stranded breaks (DSBs) in the DNA. However, DSBs are lethal to bacterial cells [26] and require repair. When induced in the bacterial genome, DSBs can be repaired via two pathways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR is the most common repair mechanism and requires the presence of another copy of the genome for DNA repair. As such, it would normally take place during replication. The other method of DSB repair, NHEJ, is found in few bacterial strains as it is dependent on the expression of Ku and Ligase D proteins (which is limited to strains such as Bacillus subtilis and Mycobacterium smegmatis) [27]. As such, since editing using CRSPR-Cas9 prefers NHEJ as the DSB repair mechanism [28], gene editing efficiencies in typical bacterial cells (including Escherichia coli) are low. It is therefore critical to evaluate alternative gene editing techniques for bacterial genome manipulation.

In order to meet the need for an efficient non-DSB editing system that does not require a donor template, our team aims to demonstrate prime editing in bacterial cells, specifically E. coli, to highlight the versatility of the prime editor. Prime editing uses a catalytically nuclease deficient Cas protein, such as nCas9 (H840A), that will nick the bacterial DNA instead of introducing DSBs. It is also able to introduce edits into the genome without the use of a donor template.

Although prime editing has been demonstrated in E. coli by Tong, et al. [29], our team hopes to similarly showcase the expression of the PE in E. coli and subsequently test the alternative RTs developed in the other parts of our team’s project.

Methodology

The team has cloned the prime editor (PE) and the traffic light reporter into the BPK764 plasmid backbone and the TLR into the lacZ pDest backbone to showcase the expression of the prime editor in E.coli. PE2 and the pegRNA was cloned into the BPK764 backbone and is flanked by the T7 promoter and T7 terminator (Figure 1a). The TLR was cloned into the lacZ pDest backbones obtained from the distributed iGEM tool kit (Parts BBa_J435320 and BBa_J435300) and is flanked by the lac promoter and the T7 terminator (Figure 1b).

Figure 1. Plasmid backbones for PE2, TLR pegRNA and TLR. Left:In the BPK764 backbone, the Cas9 gene (dark red) was replaced with PE2 while the gRNA scaffold (blue) was replaced with the TLR pegRNA to yield a 10 kb plasmid. Right:In the lacZ pDest backbone, the lacZα gene (dark red) was replaced with TLR to yield a 5 kb plasmid. Two versions of the backbone, AE lacZ pDest (BBa_J435300) or AF lacZ pDest (BBa_J435320), exist with no differences between the main features of the plasmids.

Stbl3™ E. coli cells were transformed with either TLR1 (containing the backbone of BBa_J435320) or TLR2 (containing the backbone of BBa_J435300) plasmids. Subsequently, they were made competent and transformed once more using the PE2 plasmid. The cells were then plated on double antibiotic (ampicillin and chloramphenicol) plates are subjected to Isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction to induce the expression of both PE2 and TLR1 using the lac operator.

Results and Discussions

The BPK764 plasmid (Addgene plasmid #65767) was used as the backbone because it has previously been shown to be capable of expressing SpCas9 and sgRNA in bacterial cells [30]. As prime editing uses similar components (namely PE2 and pegRNA), it is likely that their expression can be done using the same backbone. On the other hand, the lacZ pDest plasmids were used to introduce an alternative antibiotic resistance to improve the screening process after double transformation. These plasmids also contain the lac operator, which can be used to control their expression since it is not known yet whether overexpression of these parts is toxic to the cell.

PE2 is used as the prime editor as it displays significantly higher gene editing efficiencies than the basic PE1 of about a 1.6- to 5-fold increase [31]. The TLR designed by our team is also used to provide a quick, yet accurate representation of prime editing in the cell through reporter analysis. It contains an eGFP fused to an mCherry with a premature stop codon in the eGFP. The pegRNA contains the template of the edit required for the eGFP to become functional, so expression of both plasmids will cause cells to exhibit green fluorescence under the condition that prime editing can be performed in bacterial cells.

Colonies were visible on the plates when subjected to antibiotic selection, indicating that the double transformation was successful, with uptake of both the PE2 and TLR1 or TLR2 plasmids. However, the plated cells do not exhibit green fluorescence regardless of IPTG induction and IPTG concentration (Figure 2). This suggests two possibilities: either prime editing is not successful, or expression of PE2 and TLR was not successful, preventing the expression of the remedied eGFP.

a	b	c
d	e	f

Figure 2. Doubly transformed E. coli cells plated on plates containing 120 μg/mL ampicillin, 25 μg/mL chloramphenicol, and varying concentrations of IPTG. (a, b, c) E. coli cells containing both PE2 and TLR1 were induced with 0 mM, 0.1 mM, and 1.0 mM of IPTG respectively. Plate a contains uninduced cells and is the negative control. No colonies were visibly green. (d, e, f)E. coli cells containing both PE2 and TLR2 were induced with 0 mM, 0.1 mM, and 1.0 mM of IPTG respectively. Plate d contains uninduced cells and is the negative control. No colonies were visibly green.

The observed results are highly likely due to the fact that there was no T7 RNA polymerase present in the cells for expression of PE2. The genome of Stbl3 does not contain the gene required for the expression of T7 RNA polymerase (which is carried by the DE3 phage) [32]. Moreover, the plasmids used do not encode for the expression of the polymerase, which further suggests the inability to express PE2 due to T7 RNA polymerase. Hence, to improve on the results, the team will be working on cloning in the constitutive promoter BBa_J23104 upstream of the lac operator in the PE2 plasmid to express the parts without the use of T7 RNA polymerase.

Future work

Further testing will be done on expressing PE2 and other alternative PEs (using different reverse transcriptases) in Stbl3.

Much like mammalian cells, our team believes that the development of a standardised reporter system for prime editing in bacteria will be useful for the evaluation of different PEs in the future.

YEAST

Introduction

Yeast is widely used in biotechnology because of their faster growth rate and simple nutrient requirements. Yeast cell lines are engineered for use in pharmaceutical [33] and biofuel industries.

There were previous attempts to do gene editing in yeast model organism, Saccharomyces cerevisiae using the CRISPR-Cas 9 system. The type 2 CRISPR-Cas 9 genome editor cleaves target DNA using the complex formed between the Cas9 protein and an engineered single guide RNA (sgRNA). This causes a double-stranded break (DSB) 3 bp upstream of the protospacer adjacent motif (PAM) sequence [34]. DSBs are repaired by either NHEJ or HDR. As such, using the CRISPR-Cas9 system with the DSBs repair mechanism in place, the Cas 9 protein and gRNA were able to be expressed simultaneously in the S.cerevisiae cells. But these plasmids showed toxicity and therefore, they must be removed immediately after editing to minimize off-target edits [35]. To address this issue, a modified CRISPR-Cas9 technology called nickase Cas9 (nCas9) has been developed that induced single-stranded nicks to the target site, which reduced the occurrence of off-target effects in S. cerevisiae cells [14]. However, this technology still has its limitation of needing a donor template.

Hence, our team aims to employ prime editing instead of the CRISPR-Cas 9 system on the model yeast organism S. cerevisiae to improve the efficiency of gene editing in yeast. Prime editing not only induces single stranded nicks but does not require a donor template as well. This will eliminate the toxicity caused by the Cas 9 plasmids and enables efficient cloning process.

Methodology

The team is currently cloning the prime editor and TLR plasmids. We are attempting to clone the prime editor (PE) into the pGADT7 plasmid backbone and the TLR pegRNA into the pGBKT7 backbone to showcase the expression of the prime editor in S. cerevisiae. PE2 was cloned into the pGADT7 backbone and is flanked by the ADH1 promoter and ADH1 terminator. The TLR pegRNA was cloned into the pGBKT7 backbone and is flanked by the ADH1 promoter and T7 terminator.

Future work

If prime editing is successful in yeast, our team hopes to develop a prime editing reporter system for yeast as well for the ease of testing various PEs.

REFERENCES

1. Scholefield, J. and P.T. Harrison, Prime editing – an update on the field. Gene Therapy, 2021. 28(7): p. 396-401.

2. Kotewicz, M.L., et al., Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucleic Acids Res, 1988. 16(1): p. 265-77.

3. Baranauskas, A., et al., Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. Protein Eng Des Sel, 2012. 25(10): p. 657-68.

4. Arezi, B. and H. Hogrefe, Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer. Nucleic Acids Res, 2009. 37(2): p. 473-81.

5. Lu, C., et al., Prime Editing: An All-Rounder for Genome Editing. Int J Mol Sci, 2022. 23(17).

6. Anzalone, A.V., et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019. 576(7785): p. 149-157.

7. Mathis, N., et al., Predicting prime editing efficiency and product purity by deep learning. Nat Biotechnol, 2023. 41(8): p. 1151-1159.

8. Grünewald, J., et al., Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat Biotechnol, 2023. 41(3): p. 337-343.

9. Certo, M.T., et al., Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods, 2011. 8(8): p. 671-6.

10. Liu, B., et al., A split prime editor with untethered reverse transcriptase and circular RNA template. Nat Biotechnol, 2022. 40(9): p. 1388-1393.

11. Gharahkhani, P., et al., Genome-wide meta-analysis identifies 127 open-angle glaucoma loci with consistent effect across ancestries. Nature Communications, 2021. 12(1): p. 1258.

12. Lee, J., et al., Recent advances in genome editing of stem cells for drug discovery and therapeutic application. Pharmacology & Therapeutics, 2020. 209: p. 107501.

13. Cassandri, M., et al., Zinc-finger proteins in health and disease. Cell Death Discovery, 2017. 3(1): p. 17071.

14. Nemudryi, A.A., et al., TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery. Acta Naturae, 2014. 6(3): p. 19-40.

15. Adli, M., The CRISPR tool kit for genome editing and beyond. Nature Communications, 2018. 9(1): p. 1911.

16. Aliouche, H. Gene Editing Techniques: ZFNs, TALENs or CRISPR? 2019; Available from: https://www.news-medical.net/life-sciences/Gene-Editing-Techniques-ZFNs-TALENs-or-CRISPR.aspx#:~:text=The%20dependence%20on%20a%20single,advantage%20over%20TALENS%20and%20ZFNs.

17. Szczesna, K. CRISPR-Cas9, TALENs and ZFNs - the battle in gene editing. 2023; Available from: https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/#:~:text=Recognition%20of%20the%20DNA%20site,genomic%20sites%20simultaneously%20(multiplexing).

18. Yeadon, J. Pros and cons of ZNFs, TALENs, and CRISPR/Cas. 2014; Available from: https://www.jax.org/news-and-insights/jax-blog/2014/march/pros-and-cons-of-znfs-talens-and-crispr-cas.

19. Fiumara, M., et al., Genotoxic effects of base and prime editing in human hematopoietic stem cells. Nature Biotechnology, 2023.

20. Habib, O., et al., Comprehensive analysis of prime editing outcomes in human embryonic stem cells. Nucleic Acids Research, 2022. 50(2): p. 1187-1197.

21. Zhang, Z., X. Bao, and C.-P. Lin, Progress and Prospects of Gene Editing in Pluripotent Stem Cells. Biomedicines, 2023. 11(8): p. 2168.

22. Carter, M. and J. Shieh, Chapter 11 - Gene Delivery Strategies, in Guide to Research Techniques in Neuroscience (Second Edition), M. Carter and J. Shieh, Editors. 2015, Academic Press: San Diego. p. 239-252.

23. Liao, J.C., et al., Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat Rev Microbiol, 2016. 14(5): p. 288-304.

24. Donnez, D., et al., Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms. Trends Biotechnol, 2009. 27(12): p. 706-13.

25. Abdullah, et al., CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants. Synth Syst Biotechnol, 2020. 5(4): p. 277-292.

26. Bennett, C.B., et al., Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. Proc Natl Acad Sci U S A, 1993. 90(12): p. 5613-7.

27. Dupuy, P., L. Sauviac, and C. Bruand, Stress-inducible NHEJ in bacteria: function in DNA repair and acquisition of heterologous DNA. Nucleic Acids Res, 2019. 47(3): p. 1335-1349.

28. Miyaoka, Y., et al., Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016. 6: p. 23549.

29. Tong, Y., et al., A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing. Nature Communications, 2021. 12(1): p. 5206.

30. Kleinstiver, B.P., et al., Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 2015. 523(7561): p. 481-5.

31. Builder, V. Vector FAQs: Why is my bacterial vector not expressing my recombinant protein? 2023; Available from: https://en.vectorbuilder.com/resources/faq/failed-bacterial-vector.html#.

32. Paddon, C.J. and J.D. Keasling, Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nature Reviews Microbiology, 2014. 12(5): p. 355-367.

33. Yang, Z. and M. Blenner, Genome editing systems across yeast species. Current Opinion in Biotechnology, 2020. 66: p. 255-266.

34. Yu, Z., et al., Episomal editing of synthetic constructs in yeast using CRISPR. bioRxiv, 2022: p. 2022.06.21.496881.

35. Sato, G. and K. Kuroda Overcoming the Limitations of CRISPR-Cas9 Systems in Saccharomyces cerevisiae: Off-Target Effects, Epigenome, and Mitochondrial Editing. Microorganisms, 2023. 11, DOI: 10.3390/microorganisms11041040.