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. 2016 Jul 2;13(7):605-12.
doi: 10.1080/15476286.2016.1185591. Epub 2016 May 10.

Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing in Rats by Inhibiting NHEJ and Using Cas9 Protein

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Free PMC article

Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing in Rats by Inhibiting NHEJ and Using Cas9 Protein

Yuanwu Ma et al. RNA Biol. .
Free PMC article

Abstract

Precise modifications such as site mutation, codon replacement, insertion or precise targeted deletion are needed for studies of accurate gene function. The CRISPR/Cas9 system has been proved as a powerful tool to generate gene knockout and knockin animals. But the homologous recombination (HR)-directed precise genetic modification mediated by CRISPR/Cas9 is relatively lower compared with nonhomologous end-joining (NHEJ) pathway and extremely expected to be improved. Here, in this study 2 strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreER(T2), respectively. Our result showed that both Scr7 and Cas9 protein can increase the precise modification.

Keywords: CRISPR; cas9; cre; dbndd1; fabp2; homologous recombination (HR); rat.

Figures

Figure 1.
Figure 1.
The Ligase IV inhibitor Scr7 enhances the efficiency of precise modification in rats at Fabp2 locus. (A) Strategy overview for the generation of Cre insertion rats at Fabp2 locus. The ‘two-cut’ strategy was used to generate Fabp2-Cre rats. The sgRNA targeting sites are shown as black arrows and sgRNA sequences are showed underlined in blue and the PAM are in red. The template donor plasmid was designed to fuse Cre-p2a fragment with the Fabp2 gene. Targeting sites in donor plasmid were mutated to avoid the cleavage recognized by sgRNAs. (B) The PCR amplification of Cas9/sgRNA-mediated Cre insertion at the endogenous Fabp2 locus. Up: PCR amplification of upstream of the insertion part including the left homologous arm and part of Cre coding sequence using Up-F/Up-R primers. The Up-F primer should be located out of the left homologous arm. Down: PCR amplification of the downstream of the insertion part including the right homologous arm and part of Cre coding sequence using Dw-F/Dw-R primers. Dw-R primer should be located out of the right homologous arm. An overlap region was contained in the 2 amplicons. Indel: PCR amplication of Cas9/gRNA induced NHEJ modification at the endogenous Fabp2 locus. The primers used for PCR amplication and sequencing were indicated in A and shown in Table S2. (C) Scr7 increased frequency of Cas9/sgRNA-mediated Cre insertion at Fabp2 locus. The Scr7 at different concentrations (0.5 μM, 1 μM, 2 μM) was added to the Cas9/sgRNA mixuture (Cas9 mRNA, sgRNAs and circular donor plasmid) for microinjection. The PCR-positive pups were counted as Cre insertion. (D) The chromatographs of Cre integration at the Fabp2 locus.
Figure 2.
Figure 2.
Generation of rats carrying CreERT2 coding sequence at endogenous Dbndd1 locus. (A) Strategy overview for the insertion of a CreERT2 coding sequence at endogenous Dbndd1. The sgRNA targeting sites are shown as black arrows and the sgRNA sequences are showed underlined in blue and the PAM in red. The template donor plasmid was designed to fuse p2a-CreERT2 to the last codon of Dbndd1 gene. Targeting sites in donor plasmid were mutated to avoid the cleavage recognized by sgRNAs. (B) The PCR amplification of Cas9/sgRNA-mediated Cre insertion at the endogenous Dbndd1 locus. Up: PCR amplification of upstream of the insertion part including the left homologous arm and part of CreERT2 coding sequence using Up-F/Up-R primers. The Up-F primer should be located out of the left homologous arm. Down: PCR amplification of the downstream of the insertion part including the right homologous arm and part of Cre coding sequence using Dw-F/Dw-R primers. Dw-R primer should be located out of the right homologous arm. Indel: PCR amplication of Cas9/gRNA induced NHEJ modification at the endogenous Dbndd1 locus. The primers used for PCR amplication and sequencing were indicated in A and shown in Table S2. (C) Purification of recombinant Cas9 protein. The Cas9 was cloned to pET28a vector and expressed in E. coli strain BL21 (DE3). Lane 1 was loaded with pET28a contained control E. coli. Lane 2 was loaded with pET28a-Cas9 expression E. coli. Lane 3 and Lane 4 are the purified Cas9 protein. (D) In vitro DNA cleavage assay to detect the purified Cas9 protein activity. Different concentration of purified Cas9 protein mixed with gRNA (100 nM) were used to digest 100 ng plasmid DNA at 37°C for 1 h. Lane P: control plasmid with Nestin sgRNA targeting site and no Cas9 protein was added. Lane 1:1 µl of Cas9 protein ordered from company (M0306S, NEB). Lane 2-6: DNA was digested with purified Cas9 protein (1 µl, 1/10 µl, 1/20 µl, 1/30 µl, 1/40 µl, respectively). (E) Increased frequency of Cas9/sgRNA-mediated CreERT2 insertion at Dbndd1 locus. The Cas9 mRNA was replaced with Cas9 protein in the Cas9/sgRNA components for zygotes microinjection. The Scr7 at the concentrations 1 μM was added to the Cas9/sgRNA mixture (Cas9 mRNA/protein, sgRNAs and circular donor plasmid) for microinjection. The pups with specific band were counted as CreERT2 insertion. (F) The chromatographs of CreERT2 integration at the Dbndd1 locus.
Figure 3.
Figure 3.
Genotyping of the F1 rats by crossing the potential founders with wild type SD rats. (A) PCR amplication of Fabp2 locus in 9 F1 pups derived from founder #12. The potential founder #12 (Fig. S3) was crossed with wild-type SD rats and determine the genotypes of the F1 to confirm the transmission of Cre insertion at Fabp2 locus. The Cre insertions were detected in 5 F1 pups (#2, #3, #5, #7, and #8). (B) PCR amplication of Fabp2 locus in 7 F1 pups derived from founder #2. The potential founder #2 (Fig. S3) was crossed with wild-type SD rats and determine the genotypes of the F1 to confirm the transmission of NHEJ induced mutation. Mutations were detected in all F1 pups for their parent is modified bi-allele. (C) PCR amplication of Dbndd1 locus in 9 F1 pups derived from founder #8. The potential founder #8 (Fig. S8) was crossed with wild type SD rats and determine the genotypes of the F1 to confirm the transmission of modifications at Fabp2 locus. The CreERT2 insertions were detected in 5 F1 pups (#2, #4, #5, #8, and #9) and NHEJ induced indels were detected in 4 F1 pups (#1, #3, #6, and #7).

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