Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

Figures

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  Figure 1. Construction and validation of plasmid vectors for CRISPR/Cas9-mediated knock-in. (A) Scheme for CRISPR/Cas9-mediated knock-in of exogenous DNA fragment in brain neurons. Donor vector contains exogenous DNA fragment flanked by homology arms. pCGSapI-sgRNA allows simultaneous expression of hCas9 and sgRNA. Donor and pCGSapI-sgRNA plasmid vectors are injected into lateral ventricles of embryos and transfected into cortical progenitor cells by in utero electroporation. CRISPR/Cas9-mediated HDR with donor vector results in knock-in of exogenous DNA fragment at a specific genomic locus and expression of fusion protein. (B) Schematic representation of β-actin cDNA, β-actin allele, donor vector, targeted allele, and EGFP-β-actin cDNA. Donor vector contains EGFP coding sequence flanked by 0.5-kb homology arms. (C) Genomic sequences and positions of two sgRNA target sites in exon 2 of β-actin gene. Arrows indicate target sequences and their direction. Arrowheads indicate the translation initiation codon of β-actin. PAM sequences are shown in red. (D) Schematic representation of pCGSapI-sgRNA and reporter plasmids. pCGSapI-β-actin-sgRNA plasmid contains expression cassettes of hCas9 and…

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  Figure 2. CRSIPR/Cas9-mediated knock-in of EGFP at β-actin locus in brain neurons. (A) Schematic representation of experimental procedure. Cortical progenitor cells are transfected with pCAG-TagRFP and pBSSK-EGFP-β-actin-donor together with pCGSapI-β-actin-sgRNA#2 or control pCGSapI-LacZ-sgRNA by in utero electroporation at E15.5. (B) Representative images of somatosensory cortex from transfected mice. Coronal brain sections were prepared at P14-16, followed by stained with Hoechst 33342. (C) High-resolution image of enclosed region in (B). (D) Percentage of EGFP-positive cells relative to TagRFP positive cells (n = 6 each). All values represent mean ± SEM. *** p < 0.001; Student’s t-test. Scare bars represent 200 μm in (B) and 50 μm in (C).

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  Figure 3. Validation of CRISPR/Cas9-mediated knock-in of EGFP at β-actin locus. (A) Schema represents EGFP donor, wild-type allele, and knock-in allele. Arrows indicate primers for nested genomic PCR. 5′ and 3′ targeting regions are amplified by nested PCR using primers b1-b4 and c1-c4, respectively. Blue bars indicate the boundaries between β-actin genome with identical sequence to homology arms and β-actin genome or EGFP coding sequence. (B) PCR amplification of 5′ targeting region using nested primers b2 and b4. (C) PCR amplification of 3′ targeting region using nested primers c2 and c4. (D–G) Representative sequence chromatograms of regions D–G in (A). (H) Validation of indel in EGFP-positive neurons. Semi-nested PCR was performed with indicated primers. Representative sequence chromatogram is shown. (I) Single-cell RT-PCR analysis of EGFP-β-actin mRNA in EGFP-positive neurons. The DNA fragments containing the junction between EGFP and β-actin were amplified with indicated nested primers. Representative sequence chromatogram is shown. (J) Single-cell RT-PCR analysis of β-actin mRNA in EGFP-positive neurons. The DNA fragments containing the sgRNA target site were amplified with…

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  Figure 4. CRISPR/Cas9-mediated indel mutations in EGFP-negative/TagRFP-positive neurons. (A) The PCR amplified DNA fragments containing the β-actin-sgRNA target site from each single neuron were analyzed by DNA sequencing. Example of sequence chromatogram is shown. In this PCR product, moderate multiple peaks appeared from middle of the sequence. (B) PCR product in (A) was cloned into plasmid and sequenced. PCR product in (A) carried two different indel alleles. (C) Indel mutations in EGFP-negative/TagRFP positive neurons. Intact wild type sequence is shown on the top. Arrowhead and arrow indicate the first ATG codon of β-actin gene and the direction of sgRNA target sequence, respectively. Slashes represent predicted cleavage site. Cell#1 carried indel mutations in both alleles (see A,B). Cell#2-4 carried indel mutations in a single allele. (D) Frequency of indel mutations detected in the eleven EGFP-negative/TagRFP-positive neurons.

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  Figure 5. EGFP knock-in at the β-actin locus at an earlier developmental stage. Cortical progenitor cells were transfected with pCAG-TagRFP and pBSSK-EGFP-β-actin-donor together with pCGSapI-β-actin-sgRNA#2 or control pCGSapI-LacZ-sgRNA by in utero electroporation at E13.5. Representative images of somatosensory cortex from transfected mice. Coronal brain sections were prepared at P4-5, followed by stained with DAPI (blue). The images were merged on the right. Scare bar represents 100 μm.

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  Figure 6. Effects of EGFP-tagged endogenous β-actin and exogenously overexpressed EGFP-β-actin on spine morphology of cortical layer 2/3 pyramidal neurons. (A) Representative images of dendritic spines of control, EGFP knock-in and exogenous EGFP-β-actin overexpressed neurons. Scale bar represents 5 μm. (B) Spine densities of basal dendrites of control, EGFP knock-in, and exogenous EGFP-β-actin overexpressed neurons (n = 20 from 3 mice, respectively). (C) Intensities of EGFP signals on dendrites of EGFP knock-in and exogenous EGFP-β-actin overexpressed neurons (n = 10 from 3 mice, respectively). CTL, control; KI, EGFP knock-in; OE, exogenous EGFP-β-actin overexpression. All values represent mean ± SEM. ***p < 0.001 and **p < 0.01, respectively; Tukey’s test (B) or Student’s t-test (C).

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  Figure 7. Effect of EGFP knock-in and exogenous EGFP-β-actin overexpression on physiological properties of cortical layer 2/3 pyramidal neurons. (A) Representative traces of firing pattern of control, EGFP knock-in, and exogenous EGFP-β-actin overexpressed neurons. Traces of membrane potentials of all the neurons were placed based on the actual value of membrane potential. Bold dashed lines on the sides represent 0 mV. (B–D) Basic electrophysiological properties of control, EGFP knock-in, and exogenous EGFP-β-actin overexpressed neurons. Resting membrane potential, action potential threshold, and input resistance of control (n = 13 neurons from 4 mice), EGFP knock-in (n = 15 from 4 mice), and exogenous EGFP-β-actin overexpressed neurons (n = 15 from 4 mice) are shown in (B–D), respectively. (E) Representative traces of EPSCs from control, EGFP knock-in, and exogenous EGFP-β-actin overexpressed neurons. (F,G) EPSCs in control, EGFP knock-in, and exogenous EGFP-β-actin overexpressed neurons. EPSCs frequency and amplitude of control (n = 9 neurons from 3 mice), EGFP knock-in (n = 11 from 3 mice), and exogenous EGFP-β-actin overexpressed neurons (n = 10 from 3 mice) are shown in (F,G),…