Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9

Abstract

Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.

Keywords: CRISPR/Cas9; Rian; imprinting; large fragment deletion; lncRNA.

Figure 1. Dual sgRNA:Cas9-mediated Rian gene mutations. (A) Schematic diagram of the 4 sgRNA target sites at the Rian gene locus. Rian exons are indicated by black rectangles. SgRNA target sites are indicated by red rectangles and the sequences are highlighted in red. PAM sites are underlined and highlighted in green. N-sgRNA1 is located between exon 7 and 8, N-sgRNA2 is on the exon 8, and the C-sgRNAs are beyond the last exon (Exon 21). F1/2/3 and R1/2/3 represent primer pairs used for PCR and the sequences can be found in Table S1. The scale bar represents 4 kb. (B) Demonstration of 23kb deletions in the Rian gene in founder animals from co-injections of Cas9 and dual sgRNAs. Four of the 25 founders showed a 23kb deletion in the Rian gene. The primers used were Rian-F1 and R2. M, DNA marker. WT, wild-type control. (C) Sequences of mutant alleles present in the 4 mutant founder animals. The PAM sites are underlined and highlighted in green; the target sequences are red; the mutations are in blue, lower case; deletions (-), insertions (+). WT, wild-type control.

Figure 2. Four sgRNA:Cas9-mediated Rian gene modification (A) Demonstration of 23kb deletions in the Rian gene in founder animals from co-injection of Cas9 and four sgRNAs. 23kb deletions were present in three of the nine founders. Primers used were Rian-F1 and R2. WT, wild-type control. W, water. M, DNA marker. (B) Sequences of mutant alleles present in the 3 mutant founder animals. The PAM sites are underlined and highlighted in green; the target sequences are in red; the mutations are in blue, lower case; deletions (-), insertions (+). WT, wild-type control.

Figure 3. The inheritance of Rian mutations and the reduction of expression of Rian transcript variants from the maternally inherited mutant allele. (A) Transmission of deletion alleles to offspring of founder animals. PCR results showed that four (#22, #29, #31, and #34) of the 7 founders transmitted mutations to their progeny, as also shown in Table 2 and Table S2. Primers used were Rian-F1 and R2 (Table S1). M, DNA marker. PC, positive founder genome control. WT, wild-type control. (B) Q-PCR analysis of the expression of Rian transcript variants in three organs from litters with mutant Rian alleles of either maternal or paternal origin. The founder mice were mated with wild-type C57BL/6J. Heterozygote Rian mutant offspring from the crosses between male wild-type C57BL/6J and female founders (#29 and #31) are named Rian^+/−(ma) while mutant offspring from female wild-type C57BL/6J and male founders (#22 and #34) are named Rian^+/−(pa). The three paired primer pairs (Pr-1, Pr-2 and Pr-3) used detected different Rian transcript variants. The results showed that transcripts from the Rian locus were reduced significantly in Rian^+/−(ma) mice, while no reduction was observed in Rian^+/−(pa) mice. Pr, primers. WT, wild-type. Rian^+/−(ma), heterozygous Rian mutant mice with a mutant allele of maternal origin. Rian^+/−(pa), heterozygous Rian mutant mice with a mutant allele of paternal origin.

Figure 4. Mutation of the Rian locus affects expression of nearby genes specifically in Rian^+/−(ma) mice. (A) Relative locations of six genes around Rian on mouse chromosome 12. The sharp corner shows the direction of transcription. The scale bar represents 100 kb. (B) The expressions of six genes flanking Rian was assayed by Q-PCR in brain and ovary from heterozygous mice. In Rian^+/−(ma) mice, expression of Dlk1 and Mirg increased in brain and Meg3, Mirg, and Dio3 increased in ovary. In Rian^+/−(pa) mice, expression of genes adjacent to Rian showed minimal changes.