Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a

Abstract

The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.

Figure 1

Hydrodynamic based retro-orbital sinus injection with the pCriMGET_9-12a system. (A) Schematic of the combination with hydrodynamic retro-obital sinus injection and the pCriMGET_9-12a system. pCriMGET_9-12a_donor and pSpCas9_Target_Syn-sgRNA, expressed as SpCas9-2A-EGFP and target-sgRNA and Syn-crRNA-TS-sgRNA, or pAsCas12a_Target_Syn-crRNA, expressed as AsCas12a-2A-EGFP and target-crRNA and Syn-crRNA-TS-crRNA, are injected from the retro-orbital sinus. The liver was corrected for genotyping and immunostaining on day 7 after injection. (B) Hydrodynamic retro-orbital sinus injection technique. (C) Representative immunofluorescence images of mouse liver sections injected with PBS, pSpCas9_Target_syn-sgRNA or pAsCas12a_Target_Syn-crRNA on one day after injection. EGFP (green), phalloidin (white) and DAPI (blue). Scale bar, 20 μm. The experiment was repeated in 4 individual mice. (D) Serum levels of ALT and AST after retro-orbital sinus injection of plasmid DNA. Day 0 as uninjected control. The experiment was repeated in 3 individual mice at each time point.

Figure 2

Precise in-frame knock-in of mCherry-LmnA using the pCriMGET_9-12a/CRISPR-Cas9 systems. (A) Targeting map of mCherry-LmnA. The LmnA sgRNA target site (blue arrow head) and sequence (blue letters), and the CRISPR-Cas9 PAM sequence (red letters) are shown. (B) Use of plasmids for mCherry-LmnA gene targeting. (C) Genotyping PCR for mCherry-LmnA knock-in liver. PCR for WT band detection was performed using LmnA-mCherry_GT001 and _GT002 primers for 5′ junction loci and LmnA-mCherry_GT006 and _GT007 primers for 3′ junction loci. PCR for KI band detection was performed using LmnA-mCherry_GT004 and _GT005 primers for 1st PCR and _GT001 and _GT003 primers for nested PCR at 5′ junction loci, and LmnA-mCherry_GT009 and _GT0010 primers for 1st PCR and _GT007 and _GT008 primers for nested PCR at 3′ junction loci (see (A)). (D) Representative immunofluorescence images of liver tissue injected with pCriMGET_9-12a_mCherry-LmnA and pSpCas9_LmnA-sgRNA or pSpCas9_LmnA_Syn-sgRNA plasmids. DAPI (blue), mCherry (magenta) and phalloidin (white). Scale bar, 20 μm. Enlarged image is shown as dashed box area. Knock-in ratio was calculated as the percentage of mCherry + nuclei among total nuclei (n > 13,000 nuclei from four individual slides). Mean ± s.d. from four individual slides. ****P < 0.001, by two-tailed Student's t-test.

Figure 3

Bicistronic H2B-mCherry reporter cassette targeting the albumin 3′ UTR locus by the pCriMGET_9-12a/CRISPR-Cas12a system. (A) Targeting map of Albumin-H2BmCherry. Alb-crRNA target site (blue arrow head) and sequence (blue letters), and CRISPR-Cas12a PAM sequence (red letters) are shown. (B) Use of plasmids for Albumin-H2BmCherry gene targeting. (C) Genotyping PCR for Alb-H2BmCherry knock-in liver. PCR for WT band detection was performed using Alb-H2BmCherry_GT001 and _GT002 primers for 5′ junction loci and Alb-H2BmCherry _GT006 and _GT007 primers for 3′ junction loci. PCR for KI band detection was performed using Alb-H2BmCherry_GT004 and _GT005 primers for 1st PCR and _GT001 and _GT003 primers for nested PCR at 5′ junction loci, and Alb-H2BmCherry _GT009 and _GT0010 primers for 1st PCR and _GT007 and _GT008 primers for nested PCR at 3′ junction loci (see (A)). (D) Representative immunofluorescence images of liver tissue injected with pCriMGET_9-12a_ Alb-H2BmCherry and pAsCas12a_Alb-crRNA or pAsCas12a_Alb_Syn-crRNA plasmids. DAPI (blue), mCherry (magenta) and phalloidin (white). Scale bar, 20 μm. Enlarged image is shown as dashed box area. Knock-in ratio was calculated as the percentage of mCherry + nuclei among total nuclei (n > 12,000 nuclei from four individual slides). Mean ± s.d. from four individual slides. ****P < 0.001, by two-tailed Student's t-test.

Figure 4

Precise in-frame knock-in of Egfr-mCherry by the pCriMGET_9-12a/CRISPR-Cas12a systems. (A) Targeting map of Egfr-mCherry. Egfr-crRNA target site (arrowhead) and sequence (blue letters), and CRISPR-Cas12a PAM sequence (red letters) are shown. (B) Use of plasmids for Egfr-mCherry gene targeting. (C) Genotyping PCR for Egfr-mCherry knock-in liver. PCR for WT band detection was performed using Egfr-mCherry _GT001 and _GT002 primers for 5′ junction loci and Egfr-mCherry _GT006 and _GT007 primers for 3′ junction loci. PCR for KI band detection was performed using Egfr-mCherry _GT004 and _GT005 primers for 1st PCR and _GT001 and _GT003 primers for nested PCR at 5′ junction loci, and Egfr-mCherry _GT009 and _GT0010 primers for 1st PCR and _GT007 and _GT008 primers for nested PCR at 3′ junction loci (see (A)) Asterisk indicates non-specific amplicon. (D) Representative immunofluorescence images of liver tissue injected with pCriMGET_9-12a_Egfr-mCherry and pAsCas12a_Egfr-crRNA or pAsCas12a_Egfr_Syn-crRNA plasmids. DAPI (blue), mCherry (magenta) and phalloidin (white). Scale bar, 20 μm. Enlarged image is shown as dashed box area. Knock-in ratio was calculated as the percentage of mCherry + cells among total cells (n > 13,000 cells from four individual slides). Mean ± s.d. from four individual slides. ****P < 0.001, by two-tailed Student's t-test.