In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
NUCLEIC ACIDS RESEARCH
2016; 44 (8)
The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.
View details for DOI 10.1093/nar/gkv1542
View details for Web of Science ID 000376389000007
View details for PubMedID 26762978
View details for PubMedCentralID PMC4856974
Using phage integrases in a site-specific dual integrase cassette exchange strategy.
Methods in molecular biology (Clifton, N.J.)
2015; 1239: 29-38
?C31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ?C31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome. This approach relied on placing a landing pad attP for Bxb1 integrase in the genome by using phiC31 integrase-mediated recombination at a genomic pseudo attP site. In this chapter, we present a protocol for using these integrases simultaneously to facilitate cassette exchange at a predefined location. This approach permits greater control and accuracy over integration. We also present a general method for using polymerase chain reaction assays to verify that the desired cassette exchange occurred successfully.
View details for DOI 10.1007/978-1-4939-1862-1_3
View details for PubMedID 25408400
- Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells PLOS ONE 2014; 9 (4)
The juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity
2012; 23 (7-8): 431-442
Despite considerable effort, the identification of genes that regulate complex multigenic traits such as obesity has proven difficult with conventional methodologies. The use of a chromosome substitution strain-based mapping strategy based on deep congenic analysis overcame many of the difficulties associated with gene discovery and led to the finding that the juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity. The effects of a mild Cntnap2 mutation on body weight were highly dependent on genetic background, as both obesity-promoting and obesity-resistant effects of Cntnap2 were observed on different genetic backgrounds. The more severe effect of complete TAG1 deficiency, by decreasing food intake, completely prevented the weight gain normally associated with high-fat-diet feeding. Together, these studies implicate two novel proteins in the regulation of diet-induced obesity. Moreover, as juxtaparanodal proteins have previously been implicated in various neurological disorders, our results suggest a potential genetic and molecular link between obesity and diseases such as autism and epilepsy.
View details for DOI 10.1007/s00335-012-9400-8
View details for Web of Science ID 000306694700004
View details for PubMedID 22752552
Site-Specific Recombinase Strategy to Create Induced Pluripotent Stem Cells Efficiently with Plasmid DNA
2011; 29 (11): 1696-1704
Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.
View details for DOI 10.1002/stem.730
View details for Web of Science ID 000296565500007
View details for PubMedID 21898697
View details for PubMedCentralID PMC3466168
Deep congenic analysis identifies many strong, context-dependent QTLs, one of which, Slc35b4, regulates obesity and glucose homeostasis
2011; 21 (7): 1065-1073
Although central to many studies of phenotypic variation and disease susceptibility, characterizing the genetic architecture of complex traits has been unexpectedly difficult. For example, most of the susceptibility genes that contribute to highly heritable conditions such as obesity and type 2 diabetes (T2D) remain to be identified despite intensive study. We took advantage of mouse models of diet-induced metabolic disease in chromosome substitution strains (CSSs) both to characterize the genetic architecture of diet-induced obesity and glucose homeostasis and to test the feasibility of gene discovery. Beginning with a survey of CSSs, followed with genetic and phenotypic analysis of congenic, subcongenic, and subsubcongenic strains, we identified a remarkable number of closely linked, phenotypically heterogeneous quantitative trait loci (QTLs) on mouse chromosome 6 that have unexpectedly large phenotypic effects. Although fine-mapping reduced the genomic intervals and gene content of these QTLs over 3000-fold, the average phenotypic effect on body weight was reduced less than threefold, highlighting the "fractal" nature of genetic architecture in mice. Despite this genetic complexity, we found evidence for 14 QTLs in only 32 recombination events in less than 3000 mice, and with an average of four genes located within the three body weight QTLs in the subsubcongenic strains. For Obrq2a1, genetic and functional studies collectively identified the solute receptor Slc35b4 as a regulator of obesity, insulin resistance, and gluconeogenesis. This work demonstrated the unique power of CSSs as a platform for studying complex genetic traits and identifying QTLs.
View details for DOI 10.1101/gr.120741.111
View details for Web of Science ID 000292298000007
View details for PubMedID 21507882
Modulation of RNA polymerase II subunit composition by ubiquitylation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (50): 19649-19654
Emerging evidence suggests that components of the ubiquitin-proteasome system are involved in the regulation of gene expression. A variety of factors, including transcriptional activators, coactivators, and histones, are controlled by ubiquitylation, but the mechanisms through which this modification can function in transcription are generally unknown. Here, we report that the Saccharomyces cerevisiae protein Asr1 is a RING finger ubiquitin-ligase that binds directly to RNA polymerase II via the carboxyl-terminal domain (CTD) of the largest subunit of the enzyme. We show that interaction of Asr1 with the CTD depends on serine-5 phosphorylation within the CTD and results in ubiquitylation of at least 2 subunits of the enzyme, Rpb1 and Rpb2. Ubiquitylation by Asr1 leads to the ejection of the Rpb4/Rpb7 heterodimer from the polymerase complex and is associated with inactivation of polymerase function. Our data demonstrate that ubiquitylation can directly alter the subunit composition of a core component of the transcriptional machinery and provide a paradigm for how ubiquitin can influence gene activity.
View details for DOI 10.1073/pnas.0809372105
View details for Web of Science ID 000261802300017
View details for PubMedID 19064926