Matthias Heidenreich

CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.

Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

KCNQ4 K⁺ channels tune mechanoreceptors for normal touch sensation in mouse and man

Mutations inactivating the potassium channel KCNQ4 (Kv7.4) lead to deafness in humans and mice. In addition to its expression in mechanosensitive hair cells of the inner ear, KCNQ4 is found in the auditory pathway and in trigeminal nuclei that convey somatosensory information. We have now detected KCNQ4 in the peripheral nerve endings of cutaneous rapidly adapting hair follicle and Meissner corpuscle mechanoreceptors from mice and humans. Electrophysiological recordings from single afferents from Kcnq4−/− mice and mice carrying a KCNQ4 mutation found in DFNA2-type monogenic dominant human hearing loss showed elevated mechanosensitivity and altered frequency response of rapidly adapting, but not of slowly adapting nor of D-hair, mechanoreceptor neurons. Human subjects from independent DFNA2 pedigrees outperformed age-matched control subjects when tested for vibrotactile acuity at low frequencies. This work describes a gene mutation that modulates touch sensitivity in mice and humans and establishes KCNQ4 as a specific molecular marker for rapidly adapting Meissner and a subset of hair follicle afferents.

Stretch-Activation of Angiotensin II Type 1a Receptors Contributes to the Myogenic Response of Mouse Mesenteric and Renal Arteries

Rationale: Vascular wall stretch is the major stimulus for the myogenic response of small arteries to pressure. The molecular mechanisms are elusive, but recent findings suggest that G protein–coupled receptors can elicit a stretch response. Objective: To determine whether angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. Methods and Results: We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, transient receptor potential channel 6 (TRPC6) channels, or several subtypes of the voltage-gated K+ (Kv7) gene family (KCNQ3, 4, or 5). We identified a mechanosensing mechanism in isolated mesenteric arteries and in the renal circulation that relies on coupling of the AT1R subtype a to a Gq/11 protein as a critical event to accomplish the myogenic response. Arterial mechanoactivation occurs after pharmacological block of AT1R and in the absence of angiotensinogen or TRPC6 channels. Activation of AT1R subtype a by osmotically induced membrane stretch suppresses an XE991-sensitive Kv channel current in patch-clamped vascular smooth muscle cells, and similar concentrations of XE991 enhance mesenteric and renal myogenic tone. Although XE991-sensitive KCNQ3, 4, and 5 channels are expressed in vascular smooth muscle cells, XE991-sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. Conclusions: Our results provide definitive evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand-independent, mechanoactivation of AT1R subtype a. The AT1R subtype a signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4, or 5 to enact vascular smooth muscle cell activation and elevated vascular resistance.

The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus

Mutations in KCNQ2 and KCNQ3 voltage-gated potassium channels lead to neonatal epilepsy as a consequence of their key role in regulating neuronal excitability. Previous studies in the brain have focused primarily on these KCNQ family members, which contribute to M-currents and afterhyperpolarization conductances in multiple brain areas. In contrast, the function of KCNQ5 (Kv7.5), which also displays widespread expression in the brain, is entirely unknown. Here, we developed mice that carry a dominant negative mutation in the KCNQ5 pore to probe whether it has a similar function as other KCNQ channels. This mutation renders KCNQ5dn-containing homomeric and heteromeric channels nonfunctional. We find that Kcnq5dn/dn mice are viable and have normal brain morphology. Furthermore, expression and neuronal localization of KCNQ2 and KCNQ3 subunits are unchanged. However, in the CA3 area of hippocampus, a region that highly expresses KCNQ5 channels, the medium and slow afterhyperpolarization currents are significantly reduced. In contrast, neither current is affected in the CA1 area of the hippocampus, a region with low KCNQ5 expression. Our results demonstrate that KCNQ5 channels contribute to the afterhyperpolarization currents in hippocampus in a cell type-specific manner.

KCNQ5 K+ channels control hippocampal synaptic inhibition and fast network oscillations

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) K(+) channels dampen neuronal excitability and their functional impairment may lead to epilepsy. Less is known about KCNQ5 (Kv7.5), which also displays wide expression in the brain. Here we show an unexpected role of KCNQ5 in dampening synaptic inhibition and shaping network synchronization in the hippocampus. KCNQ5 localizes to the postsynaptic site of inhibitory synapses on pyramidal cells and in interneurons. Kcnq5(dn/dn) mice lacking functional KCNQ5 channels display increased excitability of different classes of interneurons, enhanced phasic and tonic inhibition, and decreased electrical shunting of inhibitory postsynaptic currents. In vivo, loss of KCNQ5 function leads to reduced fast (gamma and ripple) hippocampal oscillations, altered gamma-rhythmic discharge of pyramidal cells and impaired spatial representations. Our work demonstrates that KCNQ5 controls excitability and function of hippocampal networks through modulation of synaptic inhibition.

Vestibular Role of KCNQ4 and KCNQ5 K+ Channels Revealed by Mouse Models

Background: KCNQ K+ channels regulate neuronal excitability, and KCNQ4 mutations cause deafness. Results: KCNQ4 and KCNQ5 expression in vestibular organ and the impact on vestibular function are investigated. Conclusion: Both channels reside in postsynaptic calyx membranes of hair cells, and loss of KCNQ4 impairs vestibular function. Significance: KCNQ4 may affect vestibular function because of a novel role in synaptic transmission. The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K+ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K+ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K+ efflux. Like the related K+ channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4−/− mice lacking KCNQ4, as well as Kcnq4dn/dn and Kcnq5dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4−/−, Kcnq5dn/dn nor Kcnq4−/−/Kcnq5dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4−/−/Kcnq5dn/dn and Kcnq4−/− mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K+ removal and modulation of synaptic transmission.

Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.

Erratum: Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array

In the version of this article initially published, in Fig. 2j, the percentage for the targets Mecp2, Nlgn3, and Drd1 should be 15.2%, not 16.9%; the same error appeared in the main text, next to last paragraph, “Our results show that ~17%...” should be “Our results show that ~15%....” In the Fig. 2 legend, KASH should be spelled out as “KASH, Klarsicht ANC1 Syne1 homology...” (not “KASH ANC1, Syne homology...”). The errors have been corrected in the HTML and PDF versions of the article.