Technical Report | Published:

CRISPR interference-based specific and efficient gene inactivation in the brain

Nature Neurosciencevolume 21pages447454 (2018) | Download Citation

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.

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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.

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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

Author notes

  1. These authors contributed equally: Yi Zheng and Wei Shen.

Affiliations

  1. State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, IDG/McGovern Institute for Brain Research, School of Life Sciences, Tsinghua University, Beijing, China

    • Yi Zheng
    • , Wei Shen
    • , Bo Yang
    • , Yao-Nan Liu
    • , Huihui Qi
    • , Xia Yu
    • , Si-Yao Lu
    • , Yun Chen
    • , Yu-Zhou Xu
    • , Yun Li
    •  & Jun Yao
  2. Key Laboratory of Genomics and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China

    • Jian Zhang
    •  & Shuangli Mi
  3. The Salk Institute for Biological Studies, Laboratory of Genetics, La Jolla, CA, USA

    • Fred H. Gage

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shuangli Mi or Jun Yao.

Integrated supplementary information

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Supplementary Figure 7

    Full-length blots presented in Figures 1 and 2.

  8. Supplementary Figure 8

    Full-length blots presented in Figures 3 and 5.

  9. Supplementary Figure 9

    Full-length blots presented in Supplementary Figures 1 and 6.

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https://doi.org/10.1038/s41593-018-0077-5