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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: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms.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

The application of inversion and deletion by CRISPR to an enhancer of the Pcdhα gene cluster reveals a new role in the regulation of the Pcdhγ cluster. (A) Single-cell Hec-1-B clone with enhancer deletion and inversion alleles obtained by CRISPR with a pair of sgRNAs. (B) Significant decreases of the Pcdh α6, α12, β3, β9, γb5, and γc3 gene expression in enhancer-deleted and inverted CRISPR cell line. (C) Enhancer deletion in F1 mice. Expression profiles of Pcdh α (D) and γ (E) clusters were measured by real-time RT–PCR using mouse brain tissues. Statistical analysis was performed by Student's t-test from three independent experiments.
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MJV016F5: The application of inversion and deletion by CRISPR to an enhancer of the Pcdhα gene cluster reveals a new role in the regulation of the Pcdhγ cluster. (A) Single-cell Hec-1-B clone with enhancer deletion and inversion alleles obtained by CRISPR with a pair of sgRNAs. (B) Significant decreases of the Pcdh α6, α12, β3, β9, γb5, and γc3 gene expression in enhancer-deleted and inverted CRISPR cell line. (C) Enhancer deletion in F1 mice. Expression profiles of Pcdh α (D) and γ (E) clusters were measured by real-time RT–PCR using mouse brain tissues. Statistical analysis was performed by Student's t-test from three independent experiments.

Mentions: To demonstrate the usefulness of the CRISPR-mediated inversions and deletions in manipulating regulatory DNA elements, we transfected Hec-1-B cells (which is triploid for the Pcdh locus) with a pair of sgRNAs for two sites flanking a DNA fragment within a known Pcdhα enhancer (Ribich et al., 2006) and screened for single-cell CRISPR clones with deletion and inversion alleles. After screening 51 single-cell clones, we obtained a CRISPR cell line with one inversion and one deletion alleles (Figure 5A and Supplementary Figure S14A). Because Hec-1-B cells express two ‘alternate isoforms’ (α6 and α12 variable genes) of the Pcdhα gene cluster (Tasic et al., 2002; Guo et al., 2012), we measured expression levels of these two Pcdhα genes in the CRISPR cell lines. Quantitative real-time RT–PCR experiments revealed a significant decrease of their expression levels compared with the wild-type (WT) control (Figure 5B). Surprisingly, the expression of the two ubiquitous isoforms of the Pcdhα cluster is significantly increased (Figure 5B), probably because the targeted element contains a NRSF/REST suppressor binding site (Kehayova et al., 2011). Interestingly, this element also regulates expression members of the Pcdh β and γ clusters (Figure 5B).Figure 5


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)

The application of inversion and deletion by CRISPR to an enhancer of the Pcdhα gene cluster reveals a new role in the regulation of the Pcdhγ cluster. (A) Single-cell Hec-1-B clone with enhancer deletion and inversion alleles obtained by CRISPR with a pair of sgRNAs. (B) Significant decreases of the Pcdh α6, α12, β3, β9, γb5, and γc3 gene expression in enhancer-deleted and inverted CRISPR cell line. (C) Enhancer deletion in F1 mice. Expression profiles of Pcdh α (D) and γ (E) clusters were measured by real-time RT–PCR using mouse brain tissues. Statistical analysis was performed by Student's t-test from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524425&req=5

MJV016F5: The application of inversion and deletion by CRISPR to an enhancer of the Pcdhα gene cluster reveals a new role in the regulation of the Pcdhγ cluster. (A) Single-cell Hec-1-B clone with enhancer deletion and inversion alleles obtained by CRISPR with a pair of sgRNAs. (B) Significant decreases of the Pcdh α6, α12, β3, β9, γb5, and γc3 gene expression in enhancer-deleted and inverted CRISPR cell line. (C) Enhancer deletion in F1 mice. Expression profiles of Pcdh α (D) and γ (E) clusters were measured by real-time RT–PCR using mouse brain tissues. Statistical analysis was performed by Student's t-test from three independent experiments.
Mentions: To demonstrate the usefulness of the CRISPR-mediated inversions and deletions in manipulating regulatory DNA elements, we transfected Hec-1-B cells (which is triploid for the Pcdh locus) with a pair of sgRNAs for two sites flanking a DNA fragment within a known Pcdhα enhancer (Ribich et al., 2006) and screened for single-cell CRISPR clones with deletion and inversion alleles. After screening 51 single-cell clones, we obtained a CRISPR cell line with one inversion and one deletion alleles (Figure 5A and Supplementary Figure S14A). Because Hec-1-B cells express two ‘alternate isoforms’ (α6 and α12 variable genes) of the Pcdhα gene cluster (Tasic et al., 2002; Guo et al., 2012), we measured expression levels of these two Pcdhα genes in the CRISPR cell lines. Quantitative real-time RT–PCR experiments revealed a significant decrease of their expression levels compared with the wild-type (WT) control (Figure 5B). Surprisingly, the expression of the two ubiquitous isoforms of the Pcdhα cluster is significantly increased (Figure 5B), probably because the targeted element contains a NRSF/REST suppressor binding site (Kehayova et al., 2011). Interestingly, this element also regulates expression members of the Pcdh β and γ clusters (Figure 5B).Figure 5

Bottom Line: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms.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