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

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.

Bi-allelic gene targeting in mouse embryonic stem cells.

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.

ZFP57 and the Targeted Maintenance of Postfertilization Genomic Imprints.

Epigenetic modifications play an important role in modulating genome function. In mammals, inappropriate epigenetic states can cause embryonic lethality and various acquired and inherited diseases; hence, it is important to understand how such states are formed and maintained in particular genomic contexts. Genomic imprinting is a process in which epigenetic states provide a sustained memory of parental origin and cause gene expression/repression from only one of the two parental chromosomes. Genomic imprinting is therefore a valuable model to decipher the principles and processes associated with the targeting and maintenance of epigenetic states in general. Krüppel-associated box zinc finger proteins (KRAB-ZFPs) are proteins that have the potential to mediate this. ZFP57, one of the best characterized proteins in this family, has been shown to target and maintain epigenetic states at imprinting control regions after fertilization. Its role in imprinting through the use of ZFP57 mutants in mouse and the wider implications of KRAB-ZFPs for the targeted maintenance of epigenetic states are discussed here.

Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and ß-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.

Efficient knockin mouse generation by ssDNA oligonucleotides and zinc-finger nuclease assisted homologous recombination in zygotes.

The generation of specific mutant animal models is critical for functional analysis of human genes. The conventional gene targeting approach in embryonic stem cells (ESCs) by homologous recombination is however laborious, slow, expensive, and limited to species with functional ESCs. It is therefore a long-sought goal to develop an efficient and simple alternative gene targeting strategy. Here we demonstrate that, by combining an efficient ZFN pair and ssODN, a restriction site and a loxP site were successfully introduced into a specific genomic locus. A targeting efficiency up to 22.22% was achieved by coinciding the insertion site and the ZFN cleavage site isogenic and keeping the length of the homology arms equal and isogenic to the endogenous target locus. Furthermore, we determine that ZFN and ssODN-assisted HR is ssODN homology arm length dependent. We further show that mutant alleles generated by ZFN and ssODN-assisted HR can be transmitted through the germline successfully. This study establishes an efficient gene targeting strategy by ZFN and ssODN-assisted HR in mouse zygotes, and provides a potential avenue for genome engineering in animal species without functional ES cell lines.

Large-scale identification of microRNA targets in murine Dgcr8-deficient embryonic stem cell lines.

Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.