Sarah Bernard Senior Thesis 2024

CRISPR Activation for SYNGAP 1 Upregulation in Haploinsufficient Mice

Sarah Bernard Senior Thesis | 2024

CRISPR Activation for SYNGAP 1 Upregulation in Haploinsufficient Mice Sarah Bernard

Introduction Neurons are the fundamental building blocks of the brain and nervous system. The brain and nervous system consists of over 100 billion neurons responsible for transmitting information through a network of interconnected cells. These specialized cells communicate at junctions called synapses, consisting of the axon terminal of the sending neuron, the synaptic cleft, and the cell body of the receiving neuron.

Information is passed from one neuron to the next using electrical or chemical impulses. Long-term potentiation (LTP) is a pivotal mechanism that underlies synaptic strength and plays a crucial role in learning and memory processes. LTP involves persistent strengthening of synaptic connections between neurons, leading to enhanced communication and increased efficacy of signal transmission. This phenomenon is often considered a cellular correlate of learning and is characterized by enduring changes in the strength and efficiency of synapses.

SYNGAP1 is a gene that plays a critical role in normal cognitive function. The gene encodes for the SYNGAP protein, which is primarily expressed in the brain, and its expression is highest in forebrain structures, including the hippocampus, cortex, and olfactory bulb.

SYNGAP regulates synaptic plasticity, neuronal homeostasis and longterm potentiation (LTP), which is important for learning and memory.

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Loss of function mutations in SYNGAP1 are a major cause of neurodevelopmental disorders. The most common loss of function mutation is SYNGAP1 haploinsufficiency, where the individual has one working copy of the SYNGAP1 gene instead of two. The working copy of the gene is unable to produce enough SYNGAP protein, resulting in disruption to neuronal function. SynGAP regulates multiple signaling cascades, with one of its one key function being that it suppresses Ras/Erk activity and limits growth-related processes including protein translation and AMPA receptor exocytosis. Reduced SynGAP protein levels cause elevated basal Ras/Erk signaling. This results in increased AMPAR surface incorporation. Increased AMPAR surface incorporation results in higher neural network excitability and enhanced excitatory 3

synaptic transmission, which disrupts the excitatory and inhibitory balance of the hippocampus and cortex.

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On the clinical side, this results in SYNGAP1-related intellectual disability (SRID). SRID makes up 2% of all intellectual disability cases and is characterized by autism, cognitive impairments, and epileptic seizures. Most patients have reduced capacity for language and are nonverbal. Patients’ IQ is usually lower than 50 and many have impaired executive functions. Seizures typically begin for SRID patients at two years old, and approximately half of people with SYNGAP1-related epilepsy have drug-resistant epilepsy.

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There are currently no clinical treatments for SYNGAP1-related intellectual disability. However, previous research has found that the restoration of SynGAP function in adult SynGAP heterozygous mice has been shown to improve SYNGAP1-haploinsufficiency related brain function and behavior.

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There are multiple hypothesized methods to increase SynGAP protein levels in adult heterozygous mice. The most common methods involve gene editing using various forms of CRISPR. In our experiment, CRISPR activation (CRISPRa) was chosen over the traditional CRISPR Cas9 gene editing. CRISPRa enables targeted activation of specific genes rather than cutting and editing them. As such, CRISPRa has a reduced risk of off-targeting modifications.

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Methods Experimental Animals: For this gene therapy experiment, a mouse model will be used to test the efficacy. A mouse model is used as the protein-coding regions of the mouse and human genomes are 85 percent identical. Rodents also reproduce quickly, have short life cycles and are easy to maintain.

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The first mouse model, designed by Johnathan Wilde, has a SYNGAP1 mouse model with a mutation modeled after a human patient with SYNGAP1 mutation.

The second knock-in mouse model is designed by the Huganir Lab, and has a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22. This frameshift mutation results in heterozygous mice that have around a 50% reduction in SYNGAP 1 protein levels, has reduced synaptic plasticity and contains key features of SYNGAP1-related intellectual disability, including hyperactivity and impaired working memory.

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Fig 1: Punnett Square showing breeding pairs of SYNGAP1 mice. XX refers to wildtype mice, while Xx refers to mice haploinsufficient in the SYNGAP1 gene, where the mice only has one working copy of the gene instead of two.

For the experiment, heterozygous mice will be bred with wildtype mice. From Figure 1, half of the offspring will be heterozygous.

Only heterozygous mice will be used to test the efficacy of the gene therapy.

Genotyping of Experimental Animals: For each mouse, a 2-3 mm ear clipping was collected, submerged in 75 μl of 50 mM NaOH, and heated in a thermocycler at 95 °C for 60 min, until the samples partially broke apart. With the collected DNA, a polymerase chain reaction (PCR) reaction was set up with the following ratios: Per reaction: 20 μl

Deionised H2O

2.5 μl

10x ThermoPol buffer

0.5 μl

10 mM dNTPs

0.5 μl

10 μM primer mix

0.1 μl

Taq DNA Polymerase

23.6 μl total In each reaction, 2 μl of DNA was added, and it was run at the following PCR program: 95 - 2 min 95 - 30 sec 56 - 30 sec 72 - 30 sec repeat steps ii, iii, iv 34x 72 - 1 min

The PCR reaction works to create amplified copies of a specific segment of DNA, so that it can be further studied or experimented on.

The steps of a PCR are as follows. First, the samples are heated until the template DNA strand is denatured into single strands. Second, primers are annealed to the two DNA strands with forward and reverse primers annealing to complementary strands. Third, the DNA polymerase enzyme extends the complementary strand by inserting new complementary nucleotides. This process is repeated 34 times.

After the PCR was run, A 2% agarose gel with SYBR DNA gel stain was made. 2μl of loading dye was added to each sample, and 20 μl from each sample was transferred to the wells in 2% agarose gel, with DNA ladder added to the last well. The gel was run at 140V for 20 min, and examined under UV light to identify the bands. For the bands that did not produce clear results, the main bands were cut out, and gel purification using the Thomas Scientific kit was performed to collect the DNA. The DNA was then sent for sequencing.

Plasmid Design:

Fig 2: Image of main plasmid before transcriptional activator was cloned in.

We chose to use CRISPR activation (CRISPRa) over the traditional CRISPR Cas9 gene editing. While the traditional CRISPRCas9 gene editing uses CAS9 to cut the DNA, CRISPRa uses deactivated Cas9, fused to a transcriptional factor which activates transcription factors in the cell and promotes gene expression by binding to specific promoter regions.

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The CRISPRa experiment was initiated by designing a main plasmid containing scrambled guide RNAs (Fig 2). When designing the main plasmid, AmpR, AmpR Promoter and Human Synapsin promoter were included. The AmpR and AmpR promoter in our plasmid allows for ampicillin resistance, and lets any gene cloned under this promoter be expressed in bacteria. The Human Synapsin promoter restricts transgene expression from an adenoviral vector exclusively to neurons. The main plasmid also contains catalytically inactive Cas9 (dCas9).

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Transcriptional factors VP64 and VPR were respectively fused to the dCas9 on the main plasmid to create two CRISPRa systems. Two different transcriptional activators, VP64 and VPR were used. VP64 enhances gene expression by recruiting elements of the basal transcription machinery to the target promoter. VPR comprises three transcription factors: VP64, P65, and RTA and has a higher expression level compared to VP64.

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Fig 3: Graph showing experimental data from George Church’s lab comparing the relative RNA expression levels of dCas9-VP64 and dCas9-VPR. There was a 10-fold and 18-fold increase in NGN2 and NEUROD1 mRNA expression levels, respectively, within dCas9-VPR cells over their VP64 counterparts, and that VPR also exhibited significantly higher transcriptional activation potential than other tested CRISPRa systems. As such, VPR was the chosen transcriptional activator for our second CRISPRa system.

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The guide RNA (gRNA) is an RNA sequence that targets a specific region of interest on the DNA strand, and directs the dCas9 nuclease there. For CRISPRa, the gRNAs target the gene’s promoter region or the transcriptional start site (TSS) to result in activation. A total of 29 different gRNAs were designed for both plasmids using CRISPOR, each targeting slightly different regions of the SYNGAP gene. The gRNAs were selected based on their specificity score and a low likelihood of out-of-frame mutations.

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Guide RNA Cloning: To test the gRNAs, the scrambled gRNA in the main plasmids were replaced with our designed gRNAs using Gibson assembly. Gibson assembly is a molecular cloning method that allows multiple DNA fragments to be joined together in a single, isothermal reaction.

For the Gibson assembly, set up the following reaction on ice: • 3 μl of DNA fragments • 10 μl Gibson Assembly Master Mix • 7 μl Deionized H2O Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at – 20°C for subsequent transformation.

The modified plasmids were then used to transform chemically competent stable 3 bacteria cells using the New England Biolabs Gibson Assembly protocol: 14

To confirm the successful integration of gRNAs into the plasmid, a colony PCR was performed on three randomly selected colonies for each gRNA. Following this, one successful colony for each gRNA was expanded overnight in LB media.

Guide RNA screening: Using the Thermo Fisher Scientific kit, miniprep was done to collect DNA from the bacterial colonies, and transfections were set up with Neuro 2A (N2A) cells. The purpose of transfection is to alter the genetic content of the host cells and change the expression of genes in the cell.

Transfection works by inserting foreign nucleic acids into the cytoplasm of cells. These nucleic acids make their way to the nucleus where they are transcribed into messenger RNA (mRNA) and affect gene expression.

In this experiment, N2A cells were plated with the respective gRNAs and transfection reagents at a concentration of 4 x 10^5 cells per well. N2A cells are mouse neuroblasts with neuronal and amoeboid stem cell morphology, and were chosen for their suitability in studying neuronal differentiation, axonal growth, and signaling pathways. The transfection was set up with 2 replicates of each gRNA, a control with 15

tdTomato. Over the subsequent three days post-transfection, cells transfected with tdTomato were monitored under fluorescence to assess transfection efficiency.

Post-transfection, RNA extraction was performed.

Complementary cDNA was synthesized through reverse transcription.

This cDNA was subsequently employed in real-time quantitative PCR (RTq-PCR), with results statistically analyzed using Log Fold change.

The isolated RNA was converted to complementary DNA (cDNA), and an RTqPCR was used to evaluate the efficacy of our gene therapy on SYNGAP upregulation.

Results and Discussion Genotyping:

Fig 4: Gel electrophoresis showing the results of mouse model genotyping. The leftmost column is DNA Electrophoresis Ladder, the second column is a water sample which acts as a negative control, and the next four columns are mouse DNA from Johnathan Wilde’s mouse line.

For Johnathan Wilde’s mouse line, instead of sending the mouse DNA for sequencing, a PCR was performed on the DNA, with the primers amplifying the segment of DNA that codes for SYNGAP1, as this could be easily done in the lab. It is expected to see a difference between bands from the SYNGAP1 heterozygous mice and for wild type mice. However, after genotyping all the mice in the colony, all of the mice were showing single bands of the same thickness, suggesting that all of the mice were wild type (Figure 4). This was abnormal as this mouse strain was previously genetically engineered to contain SYNGAP1 haploinsufficiency. As such, DNA sequencing was done. The received DNA sequence was then cross checked against the expected DNA sequence on Snapgene. The sequencing results of this mouse line showed that the original knock-in mutations were no longer present in

their genome, and that there were unintended point mutations. While the reasons for this are unknown, it could have been due to poor mouse line maintenance or accidental errors in setting up breeding pairs.

Next Steps in Mouse Line: As such, arrangements were made with the Huganir Lab, to have heterozygous mice from their knock-in mouse model to be sent. Two adult male heterozygous mice will be sent to the Feng Lab. Before any experimental procedure involving the mice can be done, the mice line must be rederived. This is a way to reduce the potential risk for transmission of pathogens from a foreign mouse when it is placed into a new housing facility. The process of mouse line rederivation works by in vitro fertilization. Sperm from the heterozygous mice will be used to fertilize an embryo from a wild type female, which will then be surgically transformed into a pathogen-free recipient female, where the litter can develop to term. This litter will then be genotyped to identify heterozygous mice that will be continued to be bred and used for testing.

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Transfection:

Fig 5: Image showing the cells transfected with tdTomato on day 3 of transfection at 10x and 20x magnification. The leftmost column shows the cells under the microscope. The middle column shows the same cells under fluorescence, where each orange dot is a cell that has begun to express tdTomato. The rightmost column shows the cells under the microscope with an overlay of the fluorescent cells.

During the process of transfection, the cells transfected with tdTomato are used to check the transfection rate as a whole. tdTomato is an orange fluorescent protein, and cells successfully transfected with tdTomato show up as fluorescent orange. The transfection rate was calculated by dividing the total number of fluorescence cells over the total number of cells in the image. When the cells transfected with tdTomato reached a 30% transfection rate, the transfection is complete.

Guide RNA testing: The final step of our gRNA testing included an RTq-PCR to check the levels of upregulation in N2A cells. The RTq-PCR is used to detect and quantify RNA. As RNA is single stranded and unstable, the RTq-PCR uses cDNA as its template. During the PCR, the amount of amplification product is measured in each cycle using fluorescence. The CT values collected from the RTq-PCR were statistically analyzed using Log Fold change to get 2 delta delta CT values, which can be used to analyze the relative changes in gene expression from real-time quantitative PCR experiments. For each test, the untransfected sample and the sample with scrambled guide RNAs were included as controls.

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For SYNGAP1 mRNA expression levels in the N2A cells, a 1520x increase is optimal. Even though the desired mRNA expression levels would be a 2x in haploinsufficient mice, expression levels decrease when the gene therapy is translated from N2A cells to a mouse model.

Fig 6: Graph showing SYNGAP1 mRNA expression levels for gRNA 1-23 with transcription factor VP64

Fig 7: Graph showing SYNGAP1 mRNA expression levels for gRNA1-6 with transcription factor VPR The expression levels for the gRNAs with transcription factor VP64 was not optimal, however gRNA 15 had the most promising results with 7x upregulation (Figure 6). Transcriptional factor VPR was used to test gRNA 1-6, and there was a higher expression level compared to when gRNA 1-6 was used with VP64. From this, it could be seen that VPR results in higher expression levels compared to VP64, which is consistent with data from George Church’s lab. Because of this, the testing of gRNA 6 was repeated with VPR to see if it would result in an optimal upregulation level.

Fig 8: Graph showing SYNGAP1 mRNA expression levels for gRNA 6 with transcription factor VP64 and VPR With the repeated gRNA screening for gRNA 6, VPR gRNA 15 had promising results with a 16x upregulation of SYNGAP, which allows us to move on to testing this gene therapy in a mouse model.

Delivery of Gene Therapy: Recombinant adeno associated viruses (rAAVs) will be used as a vector for gene therapy delivery. rAAVs are a protein-based nanoparticle engineered to traverse the cell membrane, where it can ultimately deliver foreign DNA into the nucleus of a cell.

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Facial vein injections or intracerebroventricular injections will be used to deliver the DNA to the mice. Both delivery methods have pros

and cons. While facial vein injections allow for much larger volumes of up to 50 μl to be injected, crossing the blood brain barrier poses a challenge. For intracerebroventricular injections, which administers AAVs via injection directly into the cerebral ventricles, the blood-brain barrier is bypassed, however only small volumes of up to 2.5 μL can be injected. Experimentation with both methods of rAAV delivery has to be done to determine the optimal delivery method.

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Behavioral Testing: Heterozygous SYNGAP1 knockout mice show increased locomotor activity, decreased social behavior, impaired reference spatial memory and impaired working spatial memory. After successful rAAV delivery into the mouse model, behavioral testing can be done to look out for improvements in behavior and test the efficiency of the gene therapy.

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For behavioral testing, there are many different tests that can be used: the forced swim test, hot plate test, open field test, light/dark transition test, elevated plus maze test can be used to characterize behavioral differences and test efficiency of the gene therapy. The current plan is to use the following three tests: the three chamber test, open field test, and the passive avoidance task

Fig 9: Image showing the setup of the three chamber test.

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The three chamber test assesses the mice’s cognition in the form of general sociability and interest in social novelty in rodent models of nervous system disorders. It is based on the principle that mice are naturally social animals and normally prefer to spend more time with other mice, and will investigate a novel intruder more than a familiar one. There are two parts to the three chamber test. The first part tests the mouse’s level of social interaction. a subject mouse is presented with the choice of spending time with either a novel mouse or a novel object. By tracking the amount of time the mouse spends in each chamber, we can measure its level of social interaction. The second part tests the mouse’s reaction to social novelty. The mouse encounters the first intruder as well as a second never-before-met intruder. By tracking the time spent in each chamber, its reaction to social novelty can be measured.

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Fig 10: Image showing the setup of an open field test. The leftmost image shows the arena the test takes place in, while the image on the right shows the predefined zones that are used for movement tracking.

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The open field test is a sensorimotor test used to determine general activity levels, gross locomotor activity, and exploration habits in mice models . The mouse is placed into a large white box and allowed to freely move about for 10 minutes while being recorded by an overhead camera. An automated tracking system then analyzes the footage for the following parameters: distance moved, velocity, and time spent in predefined zones as seen in Figure 10. Mice display a natural aversion to brightly lit open areas. However, they also have a drive to explore new stimuli. A less anxious mouse will show an increased exploratory behavior. A more anxious mouse will have a preference to stay close to the walls of the field. By tracking the amount of time the mouse spends walking around the outer edge of the box vs. the center of the box, we are able to make conclusions about the anxiety level of the mouse.

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Fig 11: Image showing the setup of the passive avoidance test.

The chamber is divided into two compartments: a bright compartment and a dark compartment, with a gate between the two.

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The T maze spontaneous alternation test is a fear-aggravated test used to evaluate learning and memory in mice. In which, mice learn to avoid an environment in which an aversive stimulus was previously delivered. On the first day of the test, the mice are allowed to explore both compartments. On the following day, a mild foot shock is delivered in one of the compartments. On the third day, their learning and memory are tested, as the mice are then placed back into the compartment where no shock was delivered. Mice with normal learning and memory will stay away from the chamber where they had previously received the foot shock.

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These three tests will be used on three different samples of mice: mice haploinsufficient for the SYNGAP1 gene, mice haploinsufficient for the SYNGAP1 gene that have been treated with the gene therapy, and wild type mice. By comparing the results of the behavioral testing, conclusions can be drawn about the efficacy of the gene therapy. An effective gene therapy would result in the behaviors of the wild type mice and treated mice to be similar.

Other Next Steps” In addition to behavioral testing, electrophysiology and imaging needs to be done. Electrophysiology explores the electrical activity of living neurons, and will be used to compare the electrical impulses in wild type and treated mice. Imaging will be combined with Immunohistochemistry to stain SYNGAP proteins in the brains of mice.

This is meant to ensure SYNGAP is being expressed in the correct regions of the brain.

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The brains of the mice will also be tested by a rtQ PCR to check the upregulation levels overall. If the gene therapy is not resulting in a high enough upregulation level, other transcription factors can be tested as well. One possible transcriptional activator that can be tested is VP64dCas9-VP64.

Importance: Our experiment focuses on creating a gene therapy that upregulates SYNGAP1 levels in mice. Once a suitable gene therapy is created, the typical procedure would be to test it on marmosets before human clinical trials, which is the ultimate goal.The hope is that a gene therapy that can upregulate SYNGAP protein expression in the brain can potentially alleviate symptoms in human patients with SYNGAP1 haploinsufficiency.

This gene therapy will also be impactful to parents and caregivers of children with SRID. A previous study done by Kate Baker at Cambridge university showed that compared with the UK general population, parents of children with intellectual disability reported significantly elevated emotional distress. While this paper focuses its research on participants in the UK, similar studies have been conducted to show how parents with a child with intellectual disability are four times more likely to suffer from depression compared to parents of children without intellectual disability. Being able to alleviate the symptoms of SRID would greatly improve the livelihood and mental well being of their caregivers.

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Other Applications: While this gene therapy is meant to treat SYNGAP1 haploinsufficiency, the use of gene therapy can also be applied to other haploinsufficient neurological disorders.

The neuron-specific transcription factor T-box brain 1 (TBR1) regulates brain development and is a causative gene of Autism Spectrum Disorder (ASD). In humans, Tbr1 haploinsufficiency Previous research has shown that Tbr1 haploinsufficiency results in defective axonal projections of amygdalar neurons and the impairment of social interaction, ultrasonic vocalization, associative memory and cognitive flexibility in mice. Huang’s research also showed that the upregulation of amygdalar neuronal activity improves the behavioral defects of Tbr1 haploinsufficient mice.

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Mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are a frequent cause of autism spectrum disorder. The CHD8 Gene is involved in many basic biological processes such as regulating RE-1 silencing transcription factor (REST). CHD8 haploinsufficiency disrupts neurodevelopmental trajectories with an accelerated and delayed generation of inhibitory and excitatory neurons, and leads to increased anxiety among other behavioral changes.

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With both Tbr1 and CHD8 haploinsufficiency, our hope is that CRISPRa gene therapy can be used to upregulate protein expression levels, resulting in improved behavioral traits.

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