Mao CZ et al. (2018), CRISPR/Cas9-mediated efficient and precise targ...

XB-ART-55020

FASEB J 2018 Jun 13;:fj201800093. doi: 10.1096/fj.201800093.

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CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

Mao CZ , Zheng L , Zhou YM , Wu HY , Xia JB , Liang CQ , Guo XF , Peng WT , Zhao H , Cai WB , Kim SK , Park KS , Cai DQ , Qi XF .

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Zhao, H., Cai, W.-B., Kim, S.-K., Park, K.-S., Cai, D.-Q., Qi, X.-F. CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

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Species referenced: Xenopus tropicalis
Genes referenced: gpm6b myh6 odc1 pam
GO keywords: CRISPR-cas system

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	Figure 1. A diagram of the 2BCas strategy and the pCMV-EGxxFP plasmid. A, B) 2BCas on the basis of the donor plasmid harboring conventional terminator induces concurrent cleavages in target locus and 2 baits. Targeted integration of the EGFP cassette into the myh6 locus in forward direction will be induced by the NHEJ pathway. C, D) 2BCas on the basis of donor plasmid harboring 3UTR of myh6 induces concurrent cleavages in the target locus and 2 baits. Targeted integration of the EGFP cassette into the myh6 locus in forward direction will be induced by the NHEJ pathway. E) Mechanism of the pCMV-EGxxFP plasmid-mediated gRNA evaluation in vitro. The 200–500 bp target DNA sequence that contained the gRNA site was cloned into MCS between the EGx and xFP fragments of the pCMV-EGxxFP plasmid. Upon the break of bait induced by the Cas9/gRNA system, homology-dependent repair will reconstitute the EGFP expression cassette.
	Figure 2. Effects of the 2BCas strategy on the targeted integration in the myh6 locus by targeting intron in X. tropicalis. A) Schematic illustration of gRNA-GGN18 targeted the intron region of the bait sequence at the myh6 locus of X. tropicalis. B, C) Schematic illustration of the p1M6B-pA (B) and p2M6B-pA (C) donor plasmids. Red arrows denote the gRNA-GGN18 targeted site. M6B denotes the bait sequence of the myh6 locus in X. tropicalis. D, E) Integrations in forward (D) and reverse (E) direction mediated by CRISPR/Cas9-gRNA, together with the p1M6B-pA donor plasmid. F–I ) Integrated events include desired cassette insertion with forward (F) and reverse (G) directions, as well as the plasmid backbone insertion with forward (H) and reverse (I ) directions. J ) Representative images of tadpoles injected with Cas9 mRNA and gRNA-GGN18, together with p1M6B-pA (1BCas) or p2M6B-pA (2BCas) donor plasmids. Wild-type (WT) tadpoles without injection were used as control. Arrowheads denote EGFP expression in heart, arrows denote EGFP in noncardiac muscles, and asterisks denote mCherry expression in eyes. Dual color indicates the autofluorescence of the gallbladder. K) Tadpoles with different phenotypes were counted and compared with the total developed ones after injection. Green bar denotes the tadpoles that were specifically expressed EGFP in muscles and mCherry in eyes, red bar denotes the tadpoles that were only specifically expressed mCherry in eyes, and blue bar denotes tadpoles without any color in muscles and eyes. Total tadpoles evaluated for each group (n) is shown above each column.
	Figure 3. Endogenous 3UTR improves the efficiency of targeted integration mediated by the 2BCas strategy in X. tropicalis. A) Schematic illustration of gRNA-GGN18 targeted the intron region of the bait sequence at the myh6 locus of X. tropicalis. B, C) Schematic illustration of p1M6B-3UTR (B) and p2M6B-3UTR (C) donor plasmids. Red arrows denote the gRNA-GGN18 targeted site. M6B denotes the bait sequence of the myh6 locus in X. tropicalis. D, E) Integrations in forward-targeted direction mediated by CRISPR/Cas9-gRNA, together with the p1M6B-3UTR (D) or p2M6B-3UTR (E) donor plasmid. F) Representative images of tadpoles that were injected with Cas9 mRNA and gRNA-GGN18, together with the p1M6B-3UTR (1BCas-3UTR) or p2M6B-3UTR (2BCas-3UTR) donor plasmids. Wild-type (WT) tadpoles without injection were used as control. Arrowheads denote EGFP expression in heart, arrows denote EGFP in noncardiac muscles, and asterisks denote mCherry expression in eyes. Dual color indicates autofluorescence of the gallbladder. G) Tadpoles with different phenotypes were counted and compared with the total ones developed after injection. Total tadpoles evaluated for each group (n) is shown above each column.
	Figure 4. Genotyping analysis of targeted integration in positive founder tadpoles. A, B) Schematics of genotyping RT-PCR primer design for forward targeted integration of the exogenous cassette induced by the 1BCas (A) or 2BCas (B) strategies. F1/R1 primer pair was used to amplify the 59 junction between the genome and exogenous DNA. F2/R2 primer pair was used to amplify the 39 junction for the 1BCas strategy, whereas F3/R3 primer pair was used to amplify the 39 junction for the 2BCas strategy. C) Representative RT-PCR assay data revealed the 59 junctions of forward targeted integrations at the myh6 locus in potential founder tadpoles that were injected with 1BCas-pA, 2BCas-pA, 1BCas-3UTR, or 2BCas-3UTR strategies. RT-PCR fragments of odc from the genome of tadpoles were used to monitor the quality of genomic DNA. D) Representative RT-PCR assay data revealed 39 junctions of forward targeted integrations at the myh6 locus in founder tadpoles that were injected with 1BCas-3UTR and 2BCas-3UTR strategies. E) DNA sequencing data revealed the 39 junctions that contained indels both in gRNA targeted region and downstream. The ratio of colons with targeted integration was shown in brackets at the top panel. For all panels, wild type (WT) sequence is shown at top with the target site in red and the PAM sequence in green. E, tadpoles with only eye-specific expression of mCherry; H, tadpoles with specific expression of EGFP in heart in addition to eye-specific expression of mCherry; M, DNA marker; EGFP+ /mCherry+ , tadpoles with specific expression of EGFP in heart in addition to eye-specific expression of mCherry; WT, wild-type tadpoles without injection.
	Figure 5. The circular donor greatly promotes the target integration for the 2BCas strategy compared with linear donor. A) The p2M6B-3UTR plasmid and linear EGFP cassette with 3UTR (L2M6B-3UTR) were injected into fertilized eggs (500 pg/egg), together with Cas9/gRNA-GGN18. The developmental morphology was evaluated 12 h after injection. Representative images showed greater toxicity of linear donor in embryos compared with donor plasmid. The top panel shows low magnification, and the lower panel shows high magnification. Asterisk denotes abnormal embryo. B) Quantification of abnormal development of embryos after injection with the p2M6B-3UTR plasmid (500 pg) and L2M6B-3UTR linear cassette (200–500 pg) in the presence of Cas9/gRNA-GGN18. C, D) p2M6B-3UTR plasmid (200 pg/egg), L2M6B-3UTR (200 pg/egg), and linear EGFP cassette with bGHpA (L2M6B-pA, 200 pg/egg) were injected into fertilized eggs, together with Cas9/gRNA-GGN18. C) Representative images of tadpoles that were injected with Cas9/gRNA-GGN18, together with p2M6B-3UTR, L2M6B-3UTR, or L2M6B-pA donors. Arrowheads denote EGFP expression in heart, arrows denote EGFP in noncardiac muscle, and asterisks denote mCherry expression in eyes. D) Tadpoles with different phenotypes were counted and compared with the total developed ones after injection. Total tadpoles evaluated for each group (n) is shown above each column.
	Figure 6. Effects of the 2BCas strategy on the targeted integration in the myh6 locus by targeting exon in the bait region. A) Schematic illustration of the bait sequence at the myh6 locus of X. tropicalis targeted by N18-T1 in the exon region. B) Schematic illustration of donor plasmids used in this study. C) Representative images of tadpoles that were injected with Cas9 mRNA and gRNA-GGN18, together with p1M6B-pA (1BCas), p2M6B-pA (2BCas), p1M6B-3UTR (1BCas-3UTR), and p2M6B-3UTR (2BCas3UTR) donor plasmids. Arrowheads denote EGFP expression in heart, and asterisks denote mCherry expression in eyes. D) Tadpoles with different phenotypes were counted and compared with the total developed ones after injection. Total tadpoles evaluated for each group (n) is shown above each column. E, F) Three founder tadpoles (EGFP+ /mCherry+ ) that were injected with 1BCas-3UTR or 2BCas-3UTR strategy were pooled for genomic DNA extraction, followed by DNA sequencing and mapping. E) Representative RT-PCR assay data revealed 59 junctions of forward targeted integrations at the myh6 locus mediated by 1BCas3UTR or 2BCas-3UTR strategy. F) DNA sequencing data revealed highly efficient 59 junctions that contained indels. For all panels, wild-type (WT) sequence is shown at top with target site in red and the PAM sequence in green. The ratio of colons containing 59 junction with indels was shown in brackets at the top panel. Con, tadpoles without injection; M, DNA marker.
	Figure 7. Effects of the 2BCas strategy on targeted integration in human cells. A) Schematic illustration of the bait sequence at the human GAPDH locus with potential gRNA-targeted sites in the last intron region. Arrows denote gRNA sites. B) Schematic illustration of donor plasmids for EGFP integration by different strategies. C) HEK293T cells were cotransfected with the pX330-gRNA1 and individual donor plasmid as shown in panel B for 48 h. Forward targeted integrationmediated expression of EGFP were then examined by fluorescence microscopy. pEGFP-N1 plasmid was used as positive control. Scale bar, 100 mm. Con denotes cells that were transfected with pX330-gRNA1 alone. D) Quantification of EGFP+ cells in p1GB-pA, p1GB-3UTR, p2GB-pA, and p2GB-3UTR groups as shown in panel C (n = 4/group). P , 0.05, P , 0.01. E, F) Quantitative expression of EGFP was analyzed by flow cytometry 48 h after transfection. E, F) Representative images (E) and quantitative analysis (F) showed that 3UTR greatly increased the integration and expression of the EGFP reporter gene. P , 0.001 (n = 3/group). G) EGFP expression was analyzed by real-time RT-PCR 48 h after cotransfection with pX330- gRNA1 and donor plasmid. P , 0.01 (n = 3/group). H) Enriched EGFP+ cells from the p2GB-3UTR group by puromycin treatment are shown. Scale bar, 20 mm. I ) DNA sequencing data from enriched EGFP+ cells as shown in panel F revealed 59 junctions that contained indels. The ratio of colons with targeted integration was shown in brackets at the top panel. For all panels, wildtype (WT) sequence is shown at the top with target site in red and the PAM sequence in green.