Limits...
A versatile reporter system for CRISPR-mediated chromosomal rearrangements.

Li Y, Park AI, Mou H, Colpan C, Bizhanova A, Akama-Garren E, Joshi N, Hendrickson EA, Feldser D, Yin H, Anderson DG, Jacks T, Weng Z, Xue W - Genome Biol. (2015)

Bottom Line: We discover diverse yet sequence-specific indels at the rearrangement fusion sites.Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks.These reporters allow mechanisms of chromosomal rearrangements to be investigated.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P. R. China. xlccalyx@gmail.com.

ABSTRACT
Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated.

No MeSH data available.


Related in: MedlinePlus

An inverted GFP reporter (iGFP) to visualize CRISPR/Cas9-mediated DNA inversion. a Schematic of iGFP. Red arrowheads indicate the Cas9 cutting sites recognized by the sgiGFP.1 and sgiGFP.2. Inversion of the GFP cassette will lead to GFP expression from the CMV promoter. PAM sequences are underlined. Red and blue color indicate sequences flanking the predicted fusion site (indicated by ‘/’). The blue sequence in the inverted plasmid will be reverse-complementary of the original sequence. b 293 T cells were co-transfected with 0.5 μg iGFP and 0.5 μg of two px330 plasmids (sgiGFP.1 + 2) and imaged 24 h later. c A PCR reaction detected inversion (primers p1 + p2) from total cellular DNA. The arrowhead indicates the expected inversion band. d Deep-sequencing identified perfect fusion and indels (insertions or deletions) at the DNA fusion sites. Purple bars in representative IGV images (two biological replicates) indicate insertions. Position indicates basepair position in the reference sequence. e Quantification of indels. VarFreq is the average of two replicates. 22 % of the reads mapped perfectly with predicted reference sequence, corresponding to precise ligation of the DNA breaks. f Two sgRNAs also induced deletion between CRISPR/Cas9 cutting sites. A PCR reaction detected deletion of the iGFP reporter (primers p1 + p3). The top bands are full length PCR products. An arrowhead indicates the expected deletion band
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4465146&req=5

Fig1: An inverted GFP reporter (iGFP) to visualize CRISPR/Cas9-mediated DNA inversion. a Schematic of iGFP. Red arrowheads indicate the Cas9 cutting sites recognized by the sgiGFP.1 and sgiGFP.2. Inversion of the GFP cassette will lead to GFP expression from the CMV promoter. PAM sequences are underlined. Red and blue color indicate sequences flanking the predicted fusion site (indicated by ‘/’). The blue sequence in the inverted plasmid will be reverse-complementary of the original sequence. b 293 T cells were co-transfected with 0.5 μg iGFP and 0.5 μg of two px330 plasmids (sgiGFP.1 + 2) and imaged 24 h later. c A PCR reaction detected inversion (primers p1 + p2) from total cellular DNA. The arrowhead indicates the expected inversion band. d Deep-sequencing identified perfect fusion and indels (insertions or deletions) at the DNA fusion sites. Purple bars in representative IGV images (two biological replicates) indicate insertions. Position indicates basepair position in the reference sequence. e Quantification of indels. VarFreq is the average of two replicates. 22 % of the reads mapped perfectly with predicted reference sequence, corresponding to precise ligation of the DNA breaks. f Two sgRNAs also induced deletion between CRISPR/Cas9 cutting sites. A PCR reaction detected deletion of the iGFP reporter (primers p1 + p3). The top bands are full length PCR products. An arrowhead indicates the expected deletion band

Mentions: To develop a reporter system for visualizing chromosomal rearrangements, we used an inverted GFP (iGFP) plasmid [13] to mimic intra-chromosomal inversion (Fig. 1a). The GFP coding region was cloned in the inverted orientation after the cytomegalovirus (CMV) immediate-early promoter, preventing the expression of the GFP protein. We hypothesized that if we introduced two CRISPR/Cas9-mediated DNA breaks flanking the approximately 1.0 kb GFP cassette, we might be able to invert the orientation of the iGFP (Fig. 1a). We designed two sgRNAs targeting the flanking sequences (Fig. 1a and Additional file 1: Table S1). Co-transfection of two pX330 [30] plasmids co-expressing Cas9 and sgRNAs (hereafter named sgiGFP.1 + 2) with the iGFP plasmid in human 293T cells indeed led to GFP expression (Fig. 1b), confirming that cells can ligate distant DNA breaks from inverted DNA fragments [21].Fig. 1


A versatile reporter system for CRISPR-mediated chromosomal rearrangements.

Li Y, Park AI, Mou H, Colpan C, Bizhanova A, Akama-Garren E, Joshi N, Hendrickson EA, Feldser D, Yin H, Anderson DG, Jacks T, Weng Z, Xue W - Genome Biol. (2015)

An inverted GFP reporter (iGFP) to visualize CRISPR/Cas9-mediated DNA inversion. a Schematic of iGFP. Red arrowheads indicate the Cas9 cutting sites recognized by the sgiGFP.1 and sgiGFP.2. Inversion of the GFP cassette will lead to GFP expression from the CMV promoter. PAM sequences are underlined. Red and blue color indicate sequences flanking the predicted fusion site (indicated by ‘/’). The blue sequence in the inverted plasmid will be reverse-complementary of the original sequence. b 293 T cells were co-transfected with 0.5 μg iGFP and 0.5 μg of two px330 plasmids (sgiGFP.1 + 2) and imaged 24 h later. c A PCR reaction detected inversion (primers p1 + p2) from total cellular DNA. The arrowhead indicates the expected inversion band. d Deep-sequencing identified perfect fusion and indels (insertions or deletions) at the DNA fusion sites. Purple bars in representative IGV images (two biological replicates) indicate insertions. Position indicates basepair position in the reference sequence. e Quantification of indels. VarFreq is the average of two replicates. 22 % of the reads mapped perfectly with predicted reference sequence, corresponding to precise ligation of the DNA breaks. f Two sgRNAs also induced deletion between CRISPR/Cas9 cutting sites. A PCR reaction detected deletion of the iGFP reporter (primers p1 + p3). The top bands are full length PCR products. An arrowhead indicates the expected deletion band
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465146&req=5

Fig1: An inverted GFP reporter (iGFP) to visualize CRISPR/Cas9-mediated DNA inversion. a Schematic of iGFP. Red arrowheads indicate the Cas9 cutting sites recognized by the sgiGFP.1 and sgiGFP.2. Inversion of the GFP cassette will lead to GFP expression from the CMV promoter. PAM sequences are underlined. Red and blue color indicate sequences flanking the predicted fusion site (indicated by ‘/’). The blue sequence in the inverted plasmid will be reverse-complementary of the original sequence. b 293 T cells were co-transfected with 0.5 μg iGFP and 0.5 μg of two px330 plasmids (sgiGFP.1 + 2) and imaged 24 h later. c A PCR reaction detected inversion (primers p1 + p2) from total cellular DNA. The arrowhead indicates the expected inversion band. d Deep-sequencing identified perfect fusion and indels (insertions or deletions) at the DNA fusion sites. Purple bars in representative IGV images (two biological replicates) indicate insertions. Position indicates basepair position in the reference sequence. e Quantification of indels. VarFreq is the average of two replicates. 22 % of the reads mapped perfectly with predicted reference sequence, corresponding to precise ligation of the DNA breaks. f Two sgRNAs also induced deletion between CRISPR/Cas9 cutting sites. A PCR reaction detected deletion of the iGFP reporter (primers p1 + p3). The top bands are full length PCR products. An arrowhead indicates the expected deletion band
Mentions: To develop a reporter system for visualizing chromosomal rearrangements, we used an inverted GFP (iGFP) plasmid [13] to mimic intra-chromosomal inversion (Fig. 1a). The GFP coding region was cloned in the inverted orientation after the cytomegalovirus (CMV) immediate-early promoter, preventing the expression of the GFP protein. We hypothesized that if we introduced two CRISPR/Cas9-mediated DNA breaks flanking the approximately 1.0 kb GFP cassette, we might be able to invert the orientation of the iGFP (Fig. 1a). We designed two sgRNAs targeting the flanking sequences (Fig. 1a and Additional file 1: Table S1). Co-transfection of two pX330 [30] plasmids co-expressing Cas9 and sgRNAs (hereafter named sgiGFP.1 + 2) with the iGFP plasmid in human 293T cells indeed led to GFP expression (Fig. 1b), confirming that cells can ligate distant DNA breaks from inverted DNA fragments [21].Fig. 1

Bottom Line: We discover diverse yet sequence-specific indels at the rearrangement fusion sites.Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks.These reporters allow mechanisms of chromosomal rearrangements to be investigated.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P. R. China. xlccalyx@gmail.com.

ABSTRACT
Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated.

No MeSH data available.


Related in: MedlinePlus