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, 9 (8), e105584
eCollection

CRISPR/Cas9-mediated Gene Knock-Down in Post-Mitotic Neurons

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CRISPR/Cas9-mediated Gene Knock-Down in Post-Mitotic Neurons

Christoph Straub et al. PLoS One.

Abstract

The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CRISPR/Cas9 mediated knock-down of NMDA receptors.
(A) Schematic domain organization of a GluN1 subunit. Red star indicates location of the CC_Grin1.1 target sequence. Circled N/C indicate N-/C-terminus respectively; NTD: N-terminal binding domain; LBD: ligand binding domain; roman numbers indicate transmembrane domains 1–3, ec/ic: extracellular/intracellular. (B) Genomic sequences of CRISPR/Cas9 targets in GluN1. Grin1 (encoding for mouse GluN1) is located on the reverse strand of chromosome 2, and the third transmembrane domain is encoded by exon 14. Target sequences are underlined, crRNA sequences are in red, PAM sequences in green. Numbers above DNA strand indicate chromosomal position. (C) Example traces of AMPAR currents (recorded at −70 mV, inward) and NMDAR currents (recorded at +40 mV, outward) from acute brain slices. From left to right: untransfected, pX330 (‘empty’ CRISPR/Cas9 without targeting sequence), CC_Grin1.1, CC_Grin1.2. Scale bars are 50 pA and 100 ms. (D) NMDAR/AMPAR current ratio for all cells. Untransfected control cells were interleaved with neighboring GFP-positive experimental cells. AMPAR current amplitude was measured as the peak inward current at −70 mV, while NMDAR current amplitude was measured 100 msec after stimulation at +40 mV, to avoid contamination with AMPARs. Bars indicate mean ± S.E.M., grey circles individual cells. Transfection with CC_Grin1.1 completely eliminated NMDAR currents (Kruskall-Wallis test, followed by Dunn’s test).
Figure 2
Figure 2. CRISPR/Cas9-mediated knock-down in postmitotic neurons.
(A) Example traces of AMPAR currents (recorded at −70 mV, inward) and NMDAR currents (recorded at +40 mV, outward) from organotypic slice cultures. Shown are examples from untransfected cells and CC_Grin1.1 transfected cells following 10 days expression (left) and 18 days expression (right). Scale bars are 20 pA (left) or 50 pA (right) and 100 ms. (B) NMDAR/AMPAR current ratio for all cells as described before. Untransfected and CC_Grin1.1 transfected cells were compared individually for different time points, using the Mann-Whitney test.

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References

    1. Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6: 507–512. - PubMed
    1. Luo H, Lin Y, Gao F, Zhang CT, Zhang R (2014) DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements. Nucleic Acids Res 42: D574–580. - PMC - PubMed
    1. Picciotto MR, Wickman K (1998) Using knockout and transgenic mice to study neurophysiology and behavior. Physiol Rev 78: 1131–1163. - PubMed
    1. Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, et al. (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87: 1317–1326. - PubMed
    1. Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64: 475–493. - PubMed

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