Abstract
CRISPR–Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency. By developing conditional CRISPRi tools targeting synaptotagmin I (Syt1), we modified the excitatory to inhibitory balance in the dentate gyrus of the mouse hippocampus and found that the dentate gyrus has distinct regulatory roles in learning and affective processes in mice. We therefore recommend CRISPRi as a useful tool for more rapid investigation of gene function in the mammalian brain.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
15 March 2018
In the version of this article initially published, the affiliation for Jian Zhang and Shuangli Mi was incomplete. In addition to the Key Laboratory of Genomics and Precision Medicine, they are also affiliated with the University of Chinese Academy of Sciences, Beijing, China. In Supplementary Fig. 1h,l, the molecular mass marker accompanying Snap25 was labeled 58 kDa; the correct value is 25 kDa. In Supplementary Fig. 9b,c, the top panel was labeled Syt1, with molecular mass markers ranging from 46 to 100 kDa; it is actually Snap25, with molecular mass markers ranging from 17 to 46 kDa. The errors have been corrected in the HTML and PDF versions of the article.
References
Kheirbek, M. A. et al. Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Neuron 77, 955–968 (2013).
Ramirez, S. et al. Activating positive memory engrams suppresses depression-like behaviour. Nature 522, 335–339 (2015).
Chen, Y. et al. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell. Stem Cell. 17, 233–244 (2015).
Bauer, D. E., Canver, M. C. & Orkin, S. H. Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9. J. Vis. Exp. 95, e52118 (2015).
Merkle, F. T. et al. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep. 11, 875–883 (2015).
Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).
Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Incontro, S., Asensio, C. S., Edwards, R. H. & Nicoll, R. A. Efficient, complete deletion of synaptic proteins using CRISPR. Neuron 83, 1051–1057 (2014).
Straub, C., Granger, A. J., Saulnier, J. L. & Sabatini, B. L. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS One 9, e105584 (2014).
Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015).
Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell. Biol. 17, 5–15 (2016).
Ma, H. et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 34, 528–530 (2016).
Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016).
Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016).
Thakore, P. I. et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).
Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Söllner, T. et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 (1993).
Südhof, T. C. & Rothman, J. E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).
Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Root, D. E., Hacohen, N., Hahn, W. C., Lander, E. S. & Sabatini, D. M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat. Methods 3, 715–719 (2006).
Groffen, A. J. et al. Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release. Science 327, 1614–1618 (2010).
Yao, J., Gaffaney, J. D., Kwon, S. E. & Chapman, E. R. Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release. Cell 147, 666–677 (2011).
Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl. Acad. Sci. USA 101, 18206–18211 (2004).
Oh, W. J., Noggle, S. A., Maddox, D. M. & Condie, B. G. The mouse vesicular inhibitory amino acid transporter gene: expression during embryogenesis, analysis of its core promoter in neural stem cells and a reconsideration of its alternate splicing. Gene 351, 39–49 (2005).
Kerr, A. M., Reisinger, E. & Jonas, P. Differential dependence of phasic transmitter release on synaptotagmin 1 at GABAergic and glutamatergic hippocampal synapses. Proc. Natl. Acad. Sci. USA 105, 15581–15586 (2008).
Jackman, S. L. & Regehr, W. G. The mechanisms and functions of synaptic facilitation. Neuron 94, 447–464 (2017).
Deneris, E. S. & Wyler, S. C. Serotonergic transcriptional networks and potential importance to mental health. Nat. Neurosci. 15, 519–527 (2012).
Pehrson, A. L. & Sanchez, C. Altered γ-aminobutyric acid neurotransmission in major depressive disorder: a critical review of the supporting evidence and the influence of serotonergic antidepressants. Drug. Des. Devel. Ther. 9, 603–624 (2015).
Perez, S. M. & Lodge, D. J. Hippocampal interneuron transplants reverse aberrant dopamine system function and behavior in a rodent model of schizophrenia. Mol. Psychiatry 18, 1193–1198 (2013).
Lewis, D. A., Hashimoto, T. & Volk, D. W. Cortical inhibitory neurons and schizophrenia. Nat. Rev. Neurosci. 6, 312–324 (2005).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).
Szczelkun, M. D. et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl. Acad. Sci. USA 111, 9798–9803 (2014).
Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014).
Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).
Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32, 677–683 (2014).
O’Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F. & Segal, D. J. A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 43, 3389–3404 (2015).
Cencic, R. et al. Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. PLoS. One 9, e109213 (2014).
Duan, J. et al. Genome-wide identification of CRISPR/Cas9 off-targets in human genome. Cell. Res. 24, 1009–1012 (2014).
Knight, S. C. et al. Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 350, 823–826 (2015).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Radzisheuskaya, A., Shlyueva, D., Müller, I. & Helin, K. Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression. Nucleic Acids Res. 44, e141 (2016).
Forrest, A. R. et al. A promoter-level mammalian expression atlas. Nature 507, 462–470 (2014).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Acknowledgements
We thank P.D. Hsu (The Salk Institute for Biological Studies, USA) and S. Shi (Memorial Sloan Kettering Cancer Center, USA) for discussion and comments. We thank J. Guan (Tsinghua University, China) for material help. We also thank all members of the Yao laboratory for assistance. This work was supported by the National Key R&D Program of China (Grant No. 2016YFA0101900, 2016YFC0903301), National Natural Science Foundation of China (Grant No. 31771482, 31471020, 31161120358), National Basic Research Program of China (Grant No. 2015CB910603), Beijing Municipal Science & Technology Commission (Grant No.Z161100002616010), the Key Research Program of the CAS (Grant No. KJZD-EW-L14), the Open Project of Key Laboratory of Genomic and Precision Medicine of the CAS, the Open Project of State Key Laboratory of Membrane Biology of China, The JPB Foundation, The Leona M. and Harry B. Helmsley Charitable Trust, Annette C. Merle-Smith, NIH grants R01MH114030 (F.H.G) and NIH U19MH106434 (F.H.G), and The G. Harold & Leila Y. Mathers Foundation.
Author information
Authors and Affiliations
Contributions
J.Y. and F.H.G. conceived the project. J.Y. and S.M. designed the experiments. Y.Z., Y.-N.L., J.Z., Y.-Z.X. and S.M. conducted ChIP experiments. Y.Z., Y.-N.L. and X.Y. conducted western blotting, qPCR and molecular biology experiments. H.Q. and B.Y. performed electrophysiological experiments and analyzed data. W.S., Y.Z., Y.C., S.-Y.L. and Y.L. conducted stereotactic infusion and immunofluorescence experiments. W.S. conducted animal behavioral experiments and analyzed data. Y.Z. and J.Y. analyzed experimental results and wrote the manuscript with input from all authors.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 CRISPRi shows prominent targeting specificity in neurons.
(a, e and i) Design of Syt1- and Snap25-targeting sgRNA variant arrays to test the binding of dCas9-KRAB to these two ‘false’ targets. The colored nucleic acids indicate mismatches. (b, f and j) ChIP-qPCR analysis of dCas9-KRAB binding to the targeting sites with the navigation of sgRNA variants (n = 3 for all groups). (c, g and k) qRT-PCR analysis of mRNA expression in neurons expressing different sgRNA variants (n = 3 for all groups). (d, h and l) Immunoblot analysis and quantification of protein expression in neurons with co-expression of sgRNA variants and dCas9-KRAB (n = 3 for all groups). In d, h and l, blots were cropped; full-length blots are presented in Supplementary Figure 9. All data were obtained from neurons from three independent cultures with similar results and shown as mean ± s.e.m. Statistical significance was assessed by Unpaired Student’s t-test. *P < 0.05. For detailed numbers and statistical analysis, see Supplementary Table 3.
Supplementary Figure 2 Lentiviral expression of CRISPRi in the DG.
(a) Distance (in mm) from bregma along the rostrocaudal axis. Scale bar, 1 mm. (b) Quantification of dCas9-KRAB-expressing DAPI+/GFP+ cells (n = 3). Data were obtained from neurons from three mice with similar results and shown as mean ± s.e.m.
Supplementary Figure 3 Neuronal subtype-specific expression of dCas9-KRAB in primary neurons.
Representative immunofluorescence images of pCaMKIIα- and pVGAT-driving dCas9-KRAB for specific expression in glutamatergic and GABAergic neurons, respectively. Scale bar, 100 μm. Data were obtained from neurons from three independent cultures with similar results.
Supplementary Figure 4 Expression and function of Syt2 and Syt9 in GABAergic hippocampal neurons.
(a) The mRNA expression of Syt1/2/9 in the pVGAT::dCas9-KRAB+ GABAergic neurons and pVGAT::dCas9-KRAB- glutamatergic neurons (n = 3 for all groups). Data were obtained from neurons from three independent cultures with similar results and shown as mean ± s.e.m. Statistical significance was assessed by Unpaired Student’s t-test. **P = 0.001. (b, c) Representative traces (b) and average peak amplitudes (c) of AP-evoked IPSCs showing functions of Syt2 and Syt9 in the pVGAT::dCas9-KRAB+ GABAergic neurons (n = 35, 28, 13, 32). Data were obtained from neurons from three independent cultures with similar results, n represents the number cells analyzed. Quantification is represented as a box-and-whisker plot with upper and lower whiskers representing the maximum and minimum values, respectively; the boxes represent 2.5%, median and 97.5% quartiles. Statistical significance was assessed by Unpaired Student’s t-test; **P < 0.001. For detailed numbers and statistical analysis, see Supplementary Table 3.
Supplementary Figure 5 Locomotor activity of mice with conditional Syt1 KD in the DG.
(a,b) Average velocity (a) and total locomotor distance (b) of conditional Syt1 KD mice in a 10 min open field test (n = 10, 10, 10 mice). All data were shown as mean ± s.e.m. Statistical significance was assessed by Unpaired Student’s t-test. For detailed numbers and statistical analysis, see Supplementary Table 3.
Supplementary Figure 6 dCas9-based Syt1 enhancement in primary neurons.
(a) Schematic diagram of dCas9-based synergistic activation mediator (dCas9-SAM) system for Syt1 activation. Compass arrowheads indicate the sgRNA targeting sites. (b,c) Immunoblot analysis (b) and quantification (c) of Syt1 protein level in neurons expressing the dCas9-SAM system (n = 3 for all groups). In b, blots were cropped; full-length blots are presented in Supplementary Figure 9. (d) qRT-PCR analysis of Syt1 enhancement by the dCas9-SAM system (n = 3 for all groups). Data were obtained from neurons from three independent cultures with similar results and shown as mean ± s.e.m. Statistical significance was assessed by Unpaired Student’s t-test. *P < 0.05. For detailed numbers and statistical analysis, see Supplementary Table 3.
Supplementary Figure 7
Full-length blots presented in Figures 1 and 2.
Supplementary Figure 8
Full-length blots presented in Figures 3 and 5.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9
Supplementary Tables
Supplementary Tables 1–3
Rights and permissions
About this article
Cite this article
Zheng, Y., Shen, W., Zhang, J. et al. CRISPR interference-based specific and efficient gene inactivation in the brain. Nat Neurosci 21, 447–454 (2018). https://doi.org/10.1038/s41593-018-0077-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41593-018-0077-5
This article is cited by
-
Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids
Nature Biotechnology (2024)
-
An efficient low cost means of biophysical gene transfection in primary cells
Scientific Reports (2024)
-
Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential
Functional & Integrative Genomics (2024)
-
A CRISPR interference strategy for gene expression silencing in multiple myeloma cell lines
Journal of Biological Engineering (2023)
-
A combinatorial code of neurexin-3 alternative splicing controls inhibitory synapses via a trans-synaptic dystroglycan signaling loop
Nature Communications (2023)
