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Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.

Li J, Shou J, Guo Y, Tang Y, Wu Y, Jia Z, Zhai Y, Chen Z, Xu Q, Wu Q - J Mol Cell Biol (2015)

Bottom Line: Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats.Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China.

No MeSH data available.


Related in: MedlinePlus

Targeted inversions of DNA fragments of different sizes in mice and in human cells. (A) Diagram of CRISPR with a pair of sgRNAs for two sites flanking a 1241-bp DNA fragment in the Pcdh locus. Shown are inversion junctions amplified by PCR from mouse blastocysts with specific primer pairs. An example of sequence chromatograms of the upstream and downstream junctions is shown. (B) Inversion in F0 founder mice genotyped by tail clipping. (C) Diagram of inversion of a 960-bp DNA fragment in the Pcdh locus. Shown are F0 inversion mice generated by CRISPR with a pair of sgRNAs. The upstream and downstream junctions of inversions were confirmed by PCR and Sanger sequencing. (D) Diagram of inversion of a 29401-bp DNA fragment. F0 inversion mice were genotyped by PCR with specific primer pairs and confirmed by Sanger sequencing. (E) Germline transmission in mice of the DNA fragment inversion induced by CRISPR with a pair of sgRNAs. Shown is the genotyping of DNA fragment inversion in F1 mice from the crossing of two founder mice. Chromatograms of the upstream and downstream junctions by Sanger sequencing confirmed the germline transmission of inversions in mice. (F) Inversion of a short Pcdh regulatory element (RE2) in human cells. Each inversion junction in human cells is confirmed by Sanger sequencing. Inversion of 709-bp (G) and 6277-bp (H) DNA fragments at the β-globin locus. (I) Inversion of an 18142-bp DNA fragment at the HoxD locus. (J) Inversion of an 80732-bp DNA fragment at the β-globin locus. (K) Inversion of a 256744-bp DNA fragment spanning the Pcdhα gene cluster. (L) Inversion of an 807480-bp DNA fragment spanning the Pcdh α, β, and γ gene clusters.
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MJV016F2: Targeted inversions of DNA fragments of different sizes in mice and in human cells. (A) Diagram of CRISPR with a pair of sgRNAs for two sites flanking a 1241-bp DNA fragment in the Pcdh locus. Shown are inversion junctions amplified by PCR from mouse blastocysts with specific primer pairs. An example of sequence chromatograms of the upstream and downstream junctions is shown. (B) Inversion in F0 founder mice genotyped by tail clipping. (C) Diagram of inversion of a 960-bp DNA fragment in the Pcdh locus. Shown are F0 inversion mice generated by CRISPR with a pair of sgRNAs. The upstream and downstream junctions of inversions were confirmed by PCR and Sanger sequencing. (D) Diagram of inversion of a 29401-bp DNA fragment. F0 inversion mice were genotyped by PCR with specific primer pairs and confirmed by Sanger sequencing. (E) Germline transmission in mice of the DNA fragment inversion induced by CRISPR with a pair of sgRNAs. Shown is the genotyping of DNA fragment inversion in F1 mice from the crossing of two founder mice. Chromatograms of the upstream and downstream junctions by Sanger sequencing confirmed the germline transmission of inversions in mice. (F) Inversion of a short Pcdh regulatory element (RE2) in human cells. Each inversion junction in human cells is confirmed by Sanger sequencing. Inversion of 709-bp (G) and 6277-bp (H) DNA fragments at the β-globin locus. (I) Inversion of an 18142-bp DNA fragment at the HoxD locus. (J) Inversion of an 80732-bp DNA fragment at the β-globin locus. (K) Inversion of a 256744-bp DNA fragment spanning the Pcdhα gene cluster. (L) Inversion of an 807480-bp DNA fragment spanning the Pcdh α, β, and γ gene clusters.

Mentions: To see whether inversions could be generated in mice by CRISPR, we co-injected a pair of sgRNAs into one-cell embryos, for two sites flanking a DNA fragment of 1241 bp, together with the Cas9 mRNA (Figure 2A). We prepared total genomic DNA from a batch of 68 blastocysts cultured in vitro. We successfully amplified a DNA fragment with the size equal to the expected upstream inversion junction. Cloning and sequencing confirmed the upstream junction of the inversion event (Figure 2A and Supplementary Figure S1A). Similarly, we amplified and confirmed the sequences of the downstream inversion junction (Figure 2A and Supplementary Figure S1A). In addition, we detected DNA fragment deletions in blastocysts with a pair of sgRNAs (Supplementary Figure S1A). Thus, CRISPR-mediated inversions with a pair of sgRNAs occur in mouse blastocysts.Figure 2


Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.

Li J, Shou J, Guo Y, Tang Y, Wu Y, Jia Z, Zhai Y, Chen Z, Xu Q, Wu Q - J Mol Cell Biol (2015)

Targeted inversions of DNA fragments of different sizes in mice and in human cells. (A) Diagram of CRISPR with a pair of sgRNAs for two sites flanking a 1241-bp DNA fragment in the Pcdh locus. Shown are inversion junctions amplified by PCR from mouse blastocysts with specific primer pairs. An example of sequence chromatograms of the upstream and downstream junctions is shown. (B) Inversion in F0 founder mice genotyped by tail clipping. (C) Diagram of inversion of a 960-bp DNA fragment in the Pcdh locus. Shown are F0 inversion mice generated by CRISPR with a pair of sgRNAs. The upstream and downstream junctions of inversions were confirmed by PCR and Sanger sequencing. (D) Diagram of inversion of a 29401-bp DNA fragment. F0 inversion mice were genotyped by PCR with specific primer pairs and confirmed by Sanger sequencing. (E) Germline transmission in mice of the DNA fragment inversion induced by CRISPR with a pair of sgRNAs. Shown is the genotyping of DNA fragment inversion in F1 mice from the crossing of two founder mice. Chromatograms of the upstream and downstream junctions by Sanger sequencing confirmed the germline transmission of inversions in mice. (F) Inversion of a short Pcdh regulatory element (RE2) in human cells. Each inversion junction in human cells is confirmed by Sanger sequencing. Inversion of 709-bp (G) and 6277-bp (H) DNA fragments at the β-globin locus. (I) Inversion of an 18142-bp DNA fragment at the HoxD locus. (J) Inversion of an 80732-bp DNA fragment at the β-globin locus. (K) Inversion of a 256744-bp DNA fragment spanning the Pcdhα gene cluster. (L) Inversion of an 807480-bp DNA fragment spanning the Pcdh α, β, and γ gene clusters.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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MJV016F2: Targeted inversions of DNA fragments of different sizes in mice and in human cells. (A) Diagram of CRISPR with a pair of sgRNAs for two sites flanking a 1241-bp DNA fragment in the Pcdh locus. Shown are inversion junctions amplified by PCR from mouse blastocysts with specific primer pairs. An example of sequence chromatograms of the upstream and downstream junctions is shown. (B) Inversion in F0 founder mice genotyped by tail clipping. (C) Diagram of inversion of a 960-bp DNA fragment in the Pcdh locus. Shown are F0 inversion mice generated by CRISPR with a pair of sgRNAs. The upstream and downstream junctions of inversions were confirmed by PCR and Sanger sequencing. (D) Diagram of inversion of a 29401-bp DNA fragment. F0 inversion mice were genotyped by PCR with specific primer pairs and confirmed by Sanger sequencing. (E) Germline transmission in mice of the DNA fragment inversion induced by CRISPR with a pair of sgRNAs. Shown is the genotyping of DNA fragment inversion in F1 mice from the crossing of two founder mice. Chromatograms of the upstream and downstream junctions by Sanger sequencing confirmed the germline transmission of inversions in mice. (F) Inversion of a short Pcdh regulatory element (RE2) in human cells. Each inversion junction in human cells is confirmed by Sanger sequencing. Inversion of 709-bp (G) and 6277-bp (H) DNA fragments at the β-globin locus. (I) Inversion of an 18142-bp DNA fragment at the HoxD locus. (J) Inversion of an 80732-bp DNA fragment at the β-globin locus. (K) Inversion of a 256744-bp DNA fragment spanning the Pcdhα gene cluster. (L) Inversion of an 807480-bp DNA fragment spanning the Pcdh α, β, and γ gene clusters.
Mentions: To see whether inversions could be generated in mice by CRISPR, we co-injected a pair of sgRNAs into one-cell embryos, for two sites flanking a DNA fragment of 1241 bp, together with the Cas9 mRNA (Figure 2A). We prepared total genomic DNA from a batch of 68 blastocysts cultured in vitro. We successfully amplified a DNA fragment with the size equal to the expected upstream inversion junction. Cloning and sequencing confirmed the upstream junction of the inversion event (Figure 2A and Supplementary Figure S1A). Similarly, we amplified and confirmed the sequences of the downstream inversion junction (Figure 2A and Supplementary Figure S1A). In addition, we detected DNA fragment deletions in blastocysts with a pair of sgRNAs (Supplementary Figure S1A). Thus, CRISPR-mediated inversions with a pair of sgRNAs occur in mouse blastocysts.Figure 2

Bottom Line: Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats.Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China.

No MeSH data available.


Related in: MedlinePlus