Abstract
Neurodevelopmental delay with involuntary movements (NEDIM, OMIM: 617493) is a severe, early onset neurological condition characterized by a delay in psychomotor development, hypotonia, and hyperkinetic involuntary movements. Heterozygous de novo mutations in the GNAO1 gene cause NEDIM. Gαo, the gene product of GNAO1, is the alpha subunit of Go, a member of the heterotrimeric Gi/o family of G-proteins. Go is found abundantly throughout the brain but the pathophysiological mechanisms linking Gαo functions to clinical manifestations of GNAO1- related disorders are still poorly understood. One of the most common mutant alleles among the GNAO1 encephalopathies is the c.626G>A or p.Arg209His (R209H) mutation. We developed heterozygous knock-in Gnao1+/R209H mutant mice using CRISPR/Cas9 methodology to assess whether a mouse model could replicate aspects of the NEDIM clinical pattern. Mice carrying the R209H mutation exhibited increased locomotor activity and a modest gait abnormality at 8-12 weeks. In contrast to mice carrying other mutations in Gnao1, the Gnao1+/R209H mice did not show enhanced seizure susceptibility. The atypical neuroleptic risperidone has shown efficacy in a patient with the R209H mutation. It also alleviated the hyperlocomotion phenotype observed in our mouse model but suppressed locomotion in WT mice as well. In this study, we show that Gnao1+/R209H mice mirror elements of the patient phenotype and respond to an approved pharmacological agent.
Highlights
Gnao1 +/R209H mice display hyperlocomotion and gait abnormalities.
Gnao1 +/R209H mice do not show enhanced seizure susceptibility as was observed in two other Gnao1 mutant mouse models
Similar to patients with GNAO1+/R209H mutations, the Gnao1+/R209H mutant mice show only a movement phenotype and no epilepsy
Hyperlocomotion of Gnao1 +/R209H mice was alleviated by risperidone
Introduction
Gαo is the alpha subunit of the heterotrimeric G-protein and is the most abundant heterotrimeric G protein in the central nervous system comprising 1% of mammalian brain membrane protein. Mutations in GNAO1, which encodes Gαo, have been linked to two distinct neurological conditions. In 2013, four children with early infantile epileptic encephalopathy were identified with mutations in GNAO1 [1]. Since then, a growing number of patients presenting with epilepsy and/or hyperkinetic movement disorders have been found to exhibit de novo mutations in GNAO1 [2, 3]. It was recognized in 2016 that some GNAO1 mutations result in movement disorders without epilepsy [4, 5]. To date there are over 70 published cases of children with mutations in GNAO1 presenting with early infantile epileptic encephalopathy (EIEE17: OMIM 615473) and/or neurodevelopmental disorder with involuntary movements (NEDIM: OMIM 617493) [1, 4-34].
More than forty pathological variants of GNAO1 have been reported. Using a cell-based biochemical signaling assay, we classified many of those Gαo variants for their ability to support inhibition of cAMP production in transfected HEK293 cells [2]. Some mutant Gαo proteins were unable to support receptor-mediated inhibition of cAMP having a loss-of-function (LOF) mechanism. The LOF mutants were associated with epilepsy [2]. In contrast, mutations resulting in enhanced cAMP inhibition (gain-of-function, GOF) or those which supported normal cAMP regulation (NF) were generally associated with movement disorders, though some patients with these mutations also have a relatively mild seizure disorder [33].
To permit mechanistic and preclinical drug testing studies, we had previously created a mouse model with a GNAO1 GOF mutation, G203R, that was identified in patients who showed both epilepsy and movement disorders [4]. As predicted, the Gnao1+/G203R (G203R) mutant model exhibited motor coordination and gait abnormalities as well as enhanced seizure susceptibility in pentylenetrazol (PTZ) kindling studies [35]. The R209H mutations is one of the most common pathogenic GNAO1 mutations [9]. Patients with de novo, heterozygous R209H mutations in GNAO1 display severe choreoathetosis and dystonia but do not present with seizures [5, 7, 12, 13, 17, 18]. Interestingly, the R209H mutation was found to have essentially normal function for cAMP inhibition in HEK 293T cells. Despite this normal function in an in vitro assay, it causes a severe form of movement disorder in patients, often requiring intensive care unit admission [7, 13]. This discrepancy between the normal functionality in vitro and its clear clinical pathology made the R209H mutation of substantial interest for in vivo physiological studies to better understand its mechanism.
We used a battery of behavioral tests to measure motor skills in heterozygous Gnao1+/R209H mutant mice as well as performing PTZ kindling studies to assess seizure susceptibility. As expected Gnao1+/R209H mice did not show enhanced seizure susceptibility to PTZ kindling studies. Male and female Gnao1+/R209H mice both displayed significant hyperactivity in an open field assessment. This finding was surprising as our mice in our previous GNAO1-related movement disorder model, Gnao1+/G203R did not show significant differences on the open field test [35]. This difference in movement phenotype is consistent with the wide heterogeneity of movement patterns displayed by patients with GNAO1 mutations[36-42].
Having a mouse model with a strong movement phenotype will facilitate mechanistic studies of GNAO1 mutants and it allowed us to begin allele-specific preclinical drug testing. The neuroleptic risperidone was reportedly beneficial in a patient with R209H [7]. Here we show that risperidone attenuates hyperactivity in our animal model. This suggests that risperidone or related agents may be beneficial for GNAO1 patients with the R209H mutation.
Experimental Procedures
Animals
Gnao1 +/R209H mice on a C57BL/6J background were generated in the MSU Transgenic and Gene Editing Facility (https://tgef.vprgs.msu.edu) as described below. Mice (8-12 weeks old) were housed on a 12-hour light/dark cycle, with ad libitum access to food and water. All experiments were performed in accordance with NIH guidelines; protocols were approved by the Michigan State University Institutional Animal Care and Use Committee.
Generation of Gnao1 R209H edited mice
Mutant Gnao1+/R209H mice were generated via CRISPR/Cas9 genome editing on a C57BL/6J genomic background. CRISPR gRNA selection and locus analysis were performed using the Benchling platform (Benchling, Inc. San Francisco, CA.). A gRNA targeting exon 6 of the Gnao1 locus (ENSMUSG00000031748) was chosen to cause a double strand break (DSB) 3bp downstream of codon R209. A single stranded oligodeoxynucleotide (ssODN) carrying the R209H mutation CGC > CAC with short homology arms was used as a repair template (Figure 1 and Table 1). Ribonucleoprotein (RNP) complexes consisting of a synthetic crRNA/tracrRNA hybrid and Alt-R® S.p. Cas9 Nuclease V3 protein (Integrated DNA Technologies, Inc. Coralville, IA), were used to deliver CRISPR components along with the ssODN to mouse zygotes via electroporation as previously described [2, 35]. Edited embryos were implanted into pseudo-pregnant dams using standard techniques. Resulting litters were screened by PCR (Phire Green HSII PCR Mastermix, F126L, Thermo Fisher, Waltham, MA.), T7 Endonuclease I assay (M0302, New England Biolabs Inc.) and Sanger sequencing (GENEWIZ, Inc. Plainfield, NJ) for edits of the target site.
Genotyping and Breeding
Studies were done on N1 R209H heterozygotes with comparisons to littermate controls. To generate Gnao1 +/R209H heterozygotes (N1 backcross), 2 founder Gnao1 +/R209H mice, 1 male and 1 female, were crossed with WT C57BL/6J mice obtained directly from The Jackson Laboratory (Bar Harbor, ME).
DNA was extracted by an alkaline method (26) from ear clips done before weaning. PCR products were generated with primers flanking the mutation site (Fwd 5’GGACAGGTGTCACAGGGGAT 3’; 5’ ACTGGCCTCCCTTGGCAATA 3’). To produce a 375 base pair (bp) product, reaction conditions were: 0.8 μl template, 4 μl 5x Promega PCR buffer, 0.4 μl 10mM dNTPs, 1 μl 10 μ M Forward Primer, 1 μl 10 μ M Reverse Primer, 0.2 μl Promega GoTaq and 12.6 μ l DNase free water (Promega catalog # M3005, Madison WI). Samples were denatured for 4 minutes at 95°C then underwent 32 cycles of PCR (95° C for 30 seconds, 63°C for 30 seconds, and 72°C for 30 seconds) followed by a 7 minute final extension at 72°C. Ethanol precipitation was done on the PCR products and then samples were sent for Sanger sequencing (GENEWIZ, Inc. Plainfield, NJ).
Behavioral Assessment
Male and female Gnao1 +/R209H mice (8-12 weeks of age) and their Gnao1 +/+ littermates underwent a battery of behavioral testing to assess motor phenotype as described previously [43]. Before each experiment, mice were acclimated for 10 minutes to the testing room. Experiments were performed by two female researchers. All behavioral studies were done by individuals who were blind to the genotype of the animals until completion of data collection.
Open Field
The open field test is frequently used to assess locomotion, exploration and anxiety [44, 45]. The test was conducted in Fusion VersaMax clear 42 cm × 42cm × 30cm arenas (Omnitech Electronics, Inc., Columbus, Ohio). Gnao1 +/R209H mice of either sex and their littermates were placed in the arena for 30 minutes. Using the Fusion Software, we evaluated distance traveled (cm) in terms of novelty, sustained, and total movement corresponding to the first 10 minutes, 10-20 minutes and total of 30 minutes. As a potential measure of anxiety, the fraction of time spent in the center was assessed. The center area was defined as the 20.32cm × 20.32cm area within the middle of the arena.
Rotarod
To assess motor skills, we used the Economex accelerating RotaRod (Columbus Instruments, Columbus OH). The protocol occurred over a two-day period. On day 1, mice were trained for three 2-min training sessions, with 10 minutes between each training trial. During the first two sessions, the rotarod maintained a constant rotational speed of 5 rpm. The third training trial started at 5 rpm and accelerated at 0.1 rpm/sec for 2 minutes. The following day, mice ran three more trials with a 10-min break in between: two more 2-min training trials and a final 5-min test trial. Each of these tests started at 5 rpm with constant acceleration of 0.1 rpm/sec. For all training and test trials, latency to fall off the spindle was recorded in seconds.
Grip Strength
To assess mouse grip strength, we used seven home-made weights (10, 18, 26, 34, 42, 49, 57 grams). The mouse was held by the middle/base of the tail and lowered to the weight. Once the mouse grasped the weighted ring with its forepaws, the mouse was lifted until the weight cleared the bench. For each weight, a mouse was given up to three trials to suspend the weight above the table for 3 seconds. If cleared, the next heaviest weight was tried. If not total time and maximum weight lifted was recorded and a grip strength score calculated from [46]. The calculated score was normalized to mouse body weight which was measured the day of the test.
DigiGait
Mouse gait analysis was performed on the DigiGait apparatus (Mouse Specifics, Inc, Framingham, MA). After acclimation, each mouse was placed on the treadmill at speeds of 18, 20, 22, 25, 28, 32, 36 cm/s. A 10-second clip was recorded with a video camera located below the belt. There was a 5-min rest between each speed. Recordings were analyzed with the DigiGait analysis program to assess the pre-specified parameters stride length and paw angle variability. Values for all four paws were averaged to give one value per mouse – the reported n values are the number of mice. In addition, the maximum speed at which each mouse was able to successfully complete a 10-second test after three attempts was recorded as described [35].
PTZ Kindling Study
A PTZ kindling protocol was performed as described [43, 47] to assess mouse susceptibility to seizure induction. Mice were injected with a sub-convulsive dose of PTZ (40mg/kg, i.p.) every other day for up to 24 days mice and were observed for 30 minutes post-dose. Kindling was defined as tonic-clonic seizures on two consecutive injection days or death after which mice were euthanized. Kaplan-Meier survival analysis was done based on the number of injections to achieve kindling.
Gαo protein expression
Mice (6-8 weeks old) were sacrificed and their brains were dissected into different regions and flash-frozen in liquid nitrogen. For Western Blot analysis, tissues were thawed on ice and homogenized for 5 min with 0.5 mm zirconium beads in a Bullet Blender (Next Advance; Troy, NY) in RIPA buffer (20mM Tris-HCl, pH7.4, 150mM NaCl, 1mM EDTA, 1mM β-glycerophospate, 1% Triton X-100 and 0.1% SDS) with protease inhibitor (Roche/1 tablet in 10 mL RIPA). Sample homogenates were centrifuged for 5 min at 4°C at 13,000 G. Supernatants were collected and protein concentrations determined using the bicinchoninic acid method (BCA method; Pierce; Rockford, IL). Protein concentration was normalized for all tissues with RIPA buffer and 2x SDS sample buffer containing β-mercaptoethanol (Sigma-Aldrich) was added. Thirty µg of protein was loaded onto a 12% Bis-Tris SDS-PAGE gel and samples were separated for 1.5 hrs at 160V. Proteins were then transferred to an Immobilon-FL PVDF membrane (Millipore, Billerica, MA) on ice either for 2 h at 100 V, 400 mA or overnight at 30V, 50mA. Immediately after transfer, PDVF membranes were washed and blocked in Odyssey PBS blocking buffer (Li-Cor) for 40 min at RT. The membranes were then incubated with anti-Gαo (rabbit; 1:1,000; sc-387; Santa Cruz biotechnologies, Santa Cruz, CA) and anti-actin (goat; 1:1,000; sc-1615; Santa Cruz) antibodies diluted in Odyssey blocking buffer with 0.1% Tween-20 overnight at 4°C. Following four 5-min washes in phosphate-buffered saline with 0.1 % Tween-20 (PBS-T), the membrane was incubated for 1 hr at room temperature with secondary antibodies (both 1:10,000; IRDye® 800CW Donkey anti-rabbit; IRDye® 680RD Donkey anti-goat; LI-COR Biosciences) diluted in Odyssey blocking buffer with 0.1 % Tween-20. The membrane was subjected to four 5 min washes in PBS-T and a final rinse in PBS for 5-min. The membrane was kept in the dark and the infrared signals at 680 and 800nm were detected with an Odyssey Fc image system (LI-COR Biosciences). The Gαo polyclonal antibody recognizes an epitope located between positions 90-140 Gαo (Santa Cruz, personal communication) so there should be no interference from the R209H mutation.
Risperidone effects on motor behavior
Naïve 8-12 week old Gnao1 +/R209H and Gnao1 +/+ littermates of either sex were tested for effects of risperidone on their activity in the open field arean. The study was run over 5 days: On day 1, mice underwent the open field protocol described above to establish a baseline. On day 3, mice were habituated in the experimental room for 10 min then given a single i.p. dose of either 2mg/kg or 0.5mg/kg risperidone (Cayman Chemical, Ann Arbor, MI) or vehicle control. Risperidone was dissolved in DMSO at a concentration of 5 mg/ml, Further dilutions were done in DI water. Thirty minutes following injection, mice were placed in the open field arena for a 30-minute testing time. On day 5, mice were retested in the same open field protocol without injection to assess drug washout.
Statistical Analysis
Data were analyzed with unpaired Students t-test or Mantel-Cox, two-way ANOVA with Bonferroni corrections as appropriate using Graphpad Prism 7.0 (GraphPad; La Jolla, CA). A p <0.05 was considered the cut off for significance throughout. Detailed discussion of statistical analyses can be found within figure legends.
Results
Gnao1+/R209H mice are produced at the expected frequency and have normal viability
Two founder Gnao1 +/R209H mice, 1 male and 1 female, were crossed with C57BL/6J mice. Out of 98 offspring of a cross of Gnao1+/R209H with WT mice, 51 heterozygotes and 47 WT were observed. Gnao1+/R209H mice exhibit no overt postural or movement abnormalities or seizures under normal housing conditions. Adult mice showed no statistically significant differences in weight between WT and Gnao1+/R209H genotypes of either sex.
Both male and female Gnao1 +/R209H are hyperactive in the Open Field arena
Patients with R209H mutation present with hyperkinetic movement disorders [5, 7, 12, 18]. To detect motor abnormalities, Gnao1 +/R209H mice were subject to a battery of behavioral tests. The open field arena was used to test overall locomotor activity. We divided the test into two sections reflecting activity in a novel environment (0-10 minutes) then sustained activity (10-30 min). Gnao1 +/R209H mice of both sexes showed markedly increased activity in both the novelty period and the sustained activity period compared to their wildtype littermates (Figure 2B). As a potential indicator of anxiety-like behavior. Male and female Gnao1+/R209H mice also displayed reduced time in center (Figure 2B).
An accelerating rotarod test was used to assess motor coordination and balance. Neither male nor female Gnao1+/R209H mice display impaired performance (Figure 2C). Also grip strength, showed no differences between Gnao1 +/R209H and wildtype littermates in either sexes (Figure 2D).
Male Gnao1+/R209H mice display a modestly reduced stride length
Gait patterns were assessed using DigiGait analysis. Male Gnao1+/R209H mice showed a highly significant genotype effect with reduced stride length compared to wildtype littermates (P<0.001, 2-way ANOVA) but the magnitude of the effect was modest 5.6% decrease averaged across all speeds. In post-test analysis though, only the top speed (36 cm/sec) reached significance individually (p<0.01). Female Gnao1+/R209H mice did not show significant differences in stride length from WT (Figure 3C & 4D). However as previously reported for G203R mutant mice, the female Gnao1+/R209H showed a significantly reduced maximum run speed on the treadmill (Figure 3E p<0.01, t-test). This was not due to reduced body size as length, detected by the digigait system (WT 9.54 cm: vs R209H 10.17 cm) and weight were not significantly different. There was no significant difference in paw angle variability for either males or females.
Gnao1 +/R209H mice are not sensitive to PTZ kindling
Repeated application of a sub-threshold convulsive stimulus, leads to the generation of full-blown convulsions [48]. GNAO1 variants differ in their ability to cause epileptic seizures in patients. Those carrying the R209H mutant allele do not exhibit a seizure disorder [5, 7, 12, 18]. In accordance with the patients’ pattern, Gnao1+/R209H mice did not show increased susceptibility to kindling-induced seizures (Figure 4 A&B). This contrasts with the previously reported increased kindling sensitivity in male G203R mutant mice [35].
Gnao1+/R209H mice have normal Gαo protein expression in the brain
To understand why R209H mutant mice do not show a phenotype difference in the kindling test while G203R mutants do, we assessed Gαo protein expression level. Cortex, hippocampus, striatum, cerebellum, brain stem and olfactory bulb were harvested and homogenized to measure the effect of the R209H mutation on Gαo protein expression. Western blots showed no difference in Gαo protein expression between WT and Gnao1+/R209H in any of the measured brain regions (Figure 5). This is consistent with our previous analysis of protein expression in HEK293T cells with transiently transfected Gαo R209H plasmid [49].
Risperidone treatment attenuated the hyperactivity of Gnao1+/R209H mice
Patients with GNAO1 mutations were tried on multiple treatments to alleviate motor symptoms, (Table S1). Risperidone, an atypical antipsychotic drug showed beneficial effects in one of the patients. It has also been effective in drug-induced dyskinesia [50]. We show that Gnao1+/R209H mice exhibit complete abrogation of movement at 2 mg/kg risperidone, which has recovered after 2 days (Figure 6A&C). WT mice also show a significant decrease in locomotion after 2 mg/kg risperidone treatment (Figure 6A). After a single 0.5mg/kg dose of risperidone both WT and Gnao1+/R209H mice exhibit a decrease in locomotion (Figure 6B). As expected, hyperactivity of mutant mice was observed during baseline testing on day 1 and the hyperactivity had returned on day 5 following the 2-day washout period after the risperidone doses (Figure 6C). Neither 2.0 mg/kg nor 0.5 mg/kg selectively affected Gnao1+/R209H as assessed by percent suppression (Supplement Figure 1).
Discussion
The goal of personalized medicine is to define treatments for an individual patient. Knowing which gene is involved is beneficial but different mutant alleles, even in the same gene, can produce quite distinct effects. Here we report a second mouse model of Gnao1-related neurological abnormalities. The Gnao1 R209H mutant mice display motor abnormalities but no seizures which differs from our previous Gnao1 G203R mouse model [35]. The present results, however, are consistent with the clinical pattern of GNAO1 R209H patients, who present with neurodevelopmental delay with involuntary movements (NEDIM) without seizures.
The phenotype of the Gnao1 R209H mutant mice were somewhat unexpected as our previous GNAO1 Mouse model, Gnao1+/G203R, showed significant motor impairments in Rotarod and Digigait but had no changes in the open field test [43]. Patients with the G203R or R209H mutations both display movement disorder with choreathetosis and dystonia as the most common [3, 4]. The patients with R209H mutants, however, have a high frequency of hypotonia [36] while this is nearly absent in patients with the G203R mutation. Also, the R209H mutant patients have somewhat more choreoathetosis than do the G203R mutant patients but similar frequencies of dystonia [36]. The striking hyperactivity of the male R209H mutant mice points to increased dopamine signaling in the striatum as a possible mechanism. The suppression of that hyperactivity with risperidone is consistent with that model.
The two movement disorder-associated GNAO1 mutations R209H and G203R also have different patterns in in vitro studies of cAMP regulation in HEK-293T cells [2]. Interestingly, the Gαo R209H mutants supports normal cAMP regulation and is expressed at normal levels both in HEK cells[49] and in the brain (this study). In contrast the G203R mutant is expressed at lower levels both in HEK cells[49] and in the mouse brain (data not shown). Despite lower expression, it signals more strongly (GOF) in the HEK cell assay.
The new Gnao1 R209H mutant mice do have a readily detectable phenotype which will be valuable in assessing the biological function. It may help explain why children with GNAO1 R209H mutations have abnormalities despite the mutant protein having essentially normal function in cAMP assays in vitro [2]. It is possible that he pathogenicity of this mutant Gαo protein arises from effects on the non-cAMP signal outputs from the Go heterotrimeric protein (e.g. ion channels, synaptic vesicle release, or neurite outgrowth).
Effective treatments are a key goal as patients with the R209H mutation experience multiple incidents of hospitalizations [7, 9, 13]. Deep brain stimulation in the globus pallidus has proven effective in GNAO1 patients in attenuating MD [5, 16, 21, 25]. However, that invasive treatment is reserved for refractory patients. Risperidone is one of the oral treatments that has proven to be beneficial, specifically in a patient with the R209H mutation [7]. Risperidone is an atypical neuroleptic, antagonizing D2 and 5-HT receptors. Gαo couples to myriad G-protein coupled receptors including the dopamine D2 receptor which is involved in movement control [51]. In our study, risperidone was able to significantly decrease the hyperlocomotion seen in our Gnao1+/R209H mouse model. At both the 0.5 mg/kg and 2.0 mg/kg dose of risperidone, hyperactivity was attenuated in our R209H mouse model. However, this response was not selective for the Gnao1+/R209H mutant mice as the WT mice also displayed a significant decrease in locomotion. This outcome suggests that risperidone treatment may be effective in repressing global movement, while not specifically targeting a Gnao1 R209H mechanism. However, as hyperkinetic movements in patients have been shown to be exacerbated under stress, illness or high temperature [7, 19], it would be interesting to see if the mouse movement phenotype were increased or induced by physical or pharmacological stress. It would be then interesting to see if risperidone treatment could target the induced movement abnormalities, potentially showing risperidone specific effects on the Gnao1 R209H mutant model.
The new mouse model described here should provide a valuable tool for future mechanistic studies of GNAO1 encephalopathies. The fact that the mouse has very distinct behavioral changes from our previous Gnao1 mutant mouse models (R209H and G184S), indicate that it is very important to consider the mutant allele as well as the mutant gene in considering genetic disorders and personalized therapies.
Declarations of interest
none
Funding
This work was supported by a grant from the Bow Foundation, Michigan State University’s department of Comparative Medicine and Integrative Biology, and MSU discretionary funding initiative.
Author Contributions
Data Curation: Casandra L. Larrivee, Huijie Feng, Elena Y. Demireva, Huirong Xie, Jeff R. Leipprandt
Formal Analysis: Casandra L. Larrivee and Huijie Feng.
Methodology: Casandra L. Larrivee, Huijie Feng, Richard R. Neubig.
Resources: Jeff R. Leipprandt.
Supervision: Casandra L. Larrivee, Huijie Feng, Richard R. Neubig.
Visualization: Huijie Feng.
Writing – original draft: Casandra L. Larrivee
Writing – review & editing: Casandra L. Larrivee, Huijie Feng, Richard R. Neubig
Acknowledgements
The authors thank Dr. Michelle Mazei-Robison for her advice on organizing the behavioral battery experiments on our mutant mouse models.
Abbreviations
- AC
- Adenylyl Cyclase
- cAMP
- Cyclic adenosine monophosphate
- EIEE17
- Early infantile epileptic encephalopathy 17
- GOF
- Gain-of-function
- GPCR
- G-protein coupled receptor
- LOF
- Loss-of-function
- NEDIM
- Neurodevelopment disorder with involuntary movements
- RNP
- Ribonucleoprotein
- tracrRNA
- Trans-activating crRNA
- crRNA
- CRISPR RNA
- ssODN
- Single-stranded oligodeoxynucleotide
- DSB
- Double strand DNA break
- PAM
- Protospacer-adjacent motif