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

Abstract

The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. 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. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. 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.

Keywords: CRISPR/Cas9; DNA regulatory element inversion; deletion; duplication; enhancer; gene cluster; genome manipulation.

Figure 1

Inversion, duplication, and deletion of a DNA fragment in the Pcdhα regulatory region by CRISPR with a pair of sgRNAs. (A) Diagram of the Pcdhα gene cluster with a DNA fragment targeted by CRISPR with two sgRNAs. The human Pcdhα gene cluster is organized into a variable region of 15 exons (13 alternate isoforms and 2 ubiquitous isoforms) followed by a constant region of three exons. The locus regulatory region is located downstream. The sgRNA-targeting sequences are underlined. The protospacer adjacent motif (PAM) sequences of NGG are highlighted in red. The positions of the forward and reverse PCR primers are indicated by arrows. (B) Inversion of the DNA fragment is indicated by a red arrowhead. Shown are the amplified upstream and downstream junctions as well as their sequences. Deleted bases are indicated by dashes. Mutated bases are shown in yellow italics. Inserted bases are also shown. (C) Duplication of the DNA fragment. The sequences at the duplication junctions are shown. (D) Deletion of the DNA fragment. PCR products at the deletion junctions are sequenced. The 25-bp inserted T-nucleotides are also shown. The efficiency of inversion, duplication, and deletion is shown under each panel.

Figure 2

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.

Figure 3

Segmental duplications by CRISPR through trans-allelic recombination in mice and human cells. (A) Diagram of CRISPR-mediated duplication by trans-allelic recombination between two DSBs in homologous chromosomes (chromatids). (B) The duplication junction (F2+R1 primer pair) as well as the entire length of segmental duplicated region (F1+R2 primer pair) were amplified from F0 mice. The segmental duplication was first confirmed by the AgeI digestion, resulting in three fragments. (C) The entire fragment of segmental duplication was confirmed by Sanger sequencing. (D) Duplication of a 6277-bp DNA fragment at the β-globin locus in human cells. Duplication junctions were identified by PCR with a pair of specific primers. An example of sequence chromatograms at the duplication junction is shown. (E) Duplication of an 80732-bp DNA fragment at the β-globin locus. (F) Duplication of a 256744-bp DNA fragment spanning the Pcdhα gene cluster. (G) Duplication of an 807480-bp DNA fragment spanning the Pcdh α, β, and γ gene clusters.

Figure 4

Combinatorial genomic inversions and duplications by CRISPR with four sgRNAs. (A) Diagram of CRISPR with four sgRNAs targeted at the Pcdh α, β, and γ gene clusters. Shown are the inversions of the Pcdh α (B), β (C), γ (D), α/β (E), β/γ (F), and α/β/γ (G) gene clusters, as well as segmental duplications of the Pcdh α (H), β (I), γ (J), α/β (K), β/γ (L), and α/β/γ (M). The amplified upstream and downstream inversion junctions or segmental duplication junctions were sequenced. An example of Sanger sequencing chromatograms for each inversion or duplication is shown.

Figure 5

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.