Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System

Hunker, Avery C.; Soden, Marta E.; Krayushkina, Dasha; Heymann, Gabriel; Awatramani, Rajeshwar; Zweifel, Larry S.

doi:10.1016/j.celrep.2020.02.092

Cited by 59 publications

(107 citation statements)

References 62 publications

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“…Freezing behavior before, during and after CS+ and CS-was scored. Generation and validation of AAV1-FLEX-SaCas9-U6-sgOprk1: The sgRNA targeted to the Oprk1 locus (sgOprk1) was designed as previously described [20]. The following oligos (Sigma) were used to clone into pAAV-FLEX-SaCas9-U6-sgRNA (Addgene 124844).…”

Section: Discussionmentioning

confidence: 99%

“…KOR potently modulates the activity of midbrain dopamine neurons [16][17][18][19]; thus, we hypothesized that KOR signaling in dopamine neurons contributes to conditioned threat generalization at high US intensity. To test this, we selectively mutated Oprk1 in dopamine-producing neurons using a single adenoassociated viral vector (AAV) CRISPR/SaCas9 system for conditional expression of SaCas9 and a single guide RNA (sgRNA) [20]. Mice expressing Cre-recombinase under control of the endogenous dopamine transporter (DAT) locus (Slc6a3-Cre, or DAT-Cre)…”

Section: Kor Signaling In Dopamine Neurons Impairs Threat Discriminationmentioning

confidence: 99%

“…Targeted deep sequencing of Oprk1 locus: AAV1-FLEX-SaCas9-sgOprk1 and AAV1-FLEX-EGFP-KASH were co-injected into the VTA of DAT-Cre mice. Nuclei isolation, FACS, and targeted deep sequencing were performed as described previously [20]. Cells were held at -70 mV and currents were evoked using a 10 sec voltage ramp from -120 mV to -50 mV.…”

Section: Surgerymentioning

confidence: 99%

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A midbrain dynorphin circuit promotes threat generalization

Zweifel

Fellinger

et al. 2020

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Discrimination between predictive and non-predictive threat stimuli decrease as threat intensity increases. The central mechanisms that mediate the transition from discriminatory to generalized threat responding remain poorly resolved. Here, we identify the stress-and dysphoria-associated kappa opioid receptor (KOR) and its ligand dynorphin (Dyn), acting in the ventral tegmental area (VTA), as a key substrate for regulating threat generalization. We identified several dynorphinergic inputs to the VTA and demonstrate that projections from the bed nucleus of the stria terminalis (BNST) and dorsal raphe nucleus (DRN) both contribute to anxiety-like behavior but differentially affect threat generalization. These data demonstrate that conditioned threat discrimination has an inverted 'U' relationship with threat intensity and establish a role for KOR/Dyn signaling in the midbrain for promoting threat generalization.

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Section: Discussionmentioning

confidence: 99%

Section: Kor Signaling In Dopamine Neurons Impairs Threat Discriminationmentioning

confidence: 99%

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A midbrain dynorphin circuit promotes threat generalization

Zweifel

Fellinger

et al. 2020

Preprint

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“…AAV Delivery: Viruses were prepared at the University of Washington according to published methods [40,41]. Four to eight weeks prior to each experiment, AgRP Cre mice (>60 days old) received bilateral ARH injections of Cre-dependent adeno-associated (AAV; serotype 1) viral vectors encoding yellow fluorescent protein, YFP (AAV-EF1α-YFP), or mCherry, mCh (AAV-EF1α-mCh), either alone or co-injected with an AAV1 designed to encode SaCas9 and a single guide RNA (sgRNA).…”

Section: Electrophysiological Solutions and Drugsmentioning

confidence: 99%

“…Generation and validation of AAV1-FLEX-SaCas9-U6-sgKcnq3: The sgRNA for targeting the Kcnq3 locus was designed as previously described [41]. The following oligos (Sigma) were used to clone into pAAV-FLEX-SaCas9-U6-sgRNA (Addgene 124844).…”

Section: Electrophysiological Solutions and Drugsmentioning

confidence: 99%

CRISPR Knockdown of Kcnq3 Attenuates the M-current in NPY/AgRP Neurons

Stincic¹,

Bosch²,

Hunker

et al. 2020

Preprint

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Arcuate nucleus Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior in response to the internal and external environment of an organism. NPY/AgRP neurons are adjacent to the median eminence, a circumventricular organ, and circulating metabolic factors and hormones communicate the energy state of the animal via these neurons by altering the excitability of NPY/AgRP neurons, which produces an appropriate change in behavior to maintain homeostasis. One example of this plasticity is seen in the M-current, a subthreshold, non-inactivating K + current that acts to modulate excitability. Fasting decreases while estradiol increases the M-current through regulation of subunit mRNA expression of Kcnq 2, 3, & 5. KCNQ2/3 heteromers are thought to mediate the majority of the M-current. Here we used a recently developed single adeno-associated viral (AAV) vector containing a recombinasedependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA against Kcnq3 to selectively delete Kcnq3 in NPY/AgRP neurons to produce a loss of function in the M-current. We found that this virus was effective at knocking down Kcnq3 but not Kcnq2 expression. With the reduced KCNQ3 channel expression NPY/AgRP neurons were more depolarized, exhibited a higher input resistance, and the rheobase current needed to induce firing was significantly reduced, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it did significantly reduce the locomotor activity as measured in open field testing. Therefore, the SaCas9-sgKcnq3 is efficient to knock down Kcnq3 expression thereby reducing the M-current and increasing the excitability of NPY/AgRP neurons.

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