DMSO increases efficiency of genome editing at two non-coding loci

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) has become the tool of choice for genome editing. Despite the fact that it has evolved as a highly efficient means to edit/replace coding sequence, CRISPR/Cas9 efficiency for “clean” editing of non-coding DNA remains low. We set out to introduce a single base-pair substitution in two intronic SNPs at the FTO locus without altering nearby non-coding sequence. Substitution efficiency increased up to 10-fold by treatment of human embryonic stem cells (ESC) with non-toxic levels of DMSO (1%) before CRISPR/Cas9 delivery. Treatment with DMSO did not result in CRISPR/Cas9 off-target effects or compromise the chromosomal stability of the ESC. Twenty-four hour treatment of human ESC with DMSO before CRISPR/Cas9 delivery may prove a simple means to increase editing efficiency of non-coding DNA without incorporation of undesirable mutations

Functional Consequences of the Human Leptin Receptor ( LEPR ) Q223R Transversion

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93715/1/oby.2008.489.pd

Associations between an Obesity Related Genetic Variant (FTO rs9939609) and Prostate Cancer Risk

Observational studies suggest that obese men have a lower risk of incident prostate cancer, but an increased risk of advanced and fatal cancers. These observations could be due to confounding, detection bias, or a biological effect of obesity. Genetic studies are less susceptible to confounding than observational epidemiology and can suggest how associations between phenotypes (such as obesity) and diseases arise. To determine whether the associations between obesity and prostate cancer are causal, we conducted a genetic association study of the relationship between a single nucleotide polymorphism known to be associated with obesity (FTO rs9939609) and prostate cancer. Data are from a population-based sample of 1550 screen-detected prostate cancers, 1815 age- and general practice matched controls with unrestricted prostate specific antigen (PSA) values and 1175 low-PSA controls (PSA <0.5 ng/ml). The rs9939609 A allele, which was associated with higher BMI in the sample, was inversely associated with overall (odds ratio (OR) versus all controls  = 0.93; 95% confidence interval (CI): 0.85–1.02 p = 0.12 per allele) and low-grade (OR = 0.90; 0.81–0.99 p = 0.03 per allele) prostate cancer risk, but positively associated with high-grade cancer among cases (OR high- versus low-grade cancer  = 1.16; 0.99–1.37 p = 0.07 per allele). Although evidence for these effects was weak, they are consistent with observational data based on BMI phenotypes and suggest that the observed association between obesity and prostate cancer is not due to confounding. Further research should confirm these findings, extend them to other BMI-related genetic variants and determine whether they are due to detection bias or obesity-related hormonal changes

Regulation of tylosin production in Streptomyces fradiae

The tylosin biosynthetic (tyl) gene cluster of S. fradiae contains at least five regulatory genes (tylP, Q, R, S, T). During promoter-probe analysis, TylP inhibited expression from tylPp in S. lividans, raising the possibility of autoregulation. TylP also negatively regulated the expression of tylQ in S. lividans, although tylQ was still switched off in a S. fradiae strain disrupted in tylP. The latter strain produced more tylosin and sporulated a day earlier, whereas over-expression of tylP resulted in reduced levels of antibiotic. Expression analysis by RT-PCR performed on the latter strain revealed barely detectable transcription from multiple genes, including tylS. This was probably a direct effect since TylP repressed tylSp in S. lividans. Meanwhile, expression analysis before and after the onset of tylosin production implied that TylQ is a key, negative regulator of tylosin production. The latter hypothesis was substantiated by expression analysis of a tylosin non-producing strain wherein tylQ was over-expressed. Moreover, expression analysis of a tylR-disrupted strain confirmed that TylQ controls tylosin production by repressing tylR. TylS, on the other hand, is essential for tylR activation and maximal expression of tylGIII-MIII, as shown by expression analysis of a strain disrupted in tylS. In the same and other genetically modified strains, expression analysis revealed that tylG and/or tylM genes are not necessarily co-transcribed or even co-regulated. This finding was supported by promoter probing in S. lividans and S. fradiae.;During an extensive portion of the empirical strain improvement program carried out at Lilly Laboratories, few changes of significance seem to have occurred within the tyl cluster. No mutations were found in the tyl promoters analyzed. Of regulatory genes, only tylQ had undergone a single point mutation that resulted in inactivation of its product. Enhancement of tylosin yields in empirically improved strains appears to have mainly occurred by mutations present outside the tyl cluster

Correctly targeted ESC clones are pluripotent.

<p>Immunofluorescence of correctly targeted ESC clone rs1421085 (C/C) showing pluripotency molecular markers homeobox transcription factor NANOG, octamer-binding homeodomain transcription factor 4 (OCT4), glycoprotein TRA-1-80, Stage-Specific Embryonic Antigen-4 (SSEA-4), and transcription factor SRY (sex determining region Y)-box 2 (SOX2). Pluripotency markers are shown in green, nuclei are counterstained with DAPI.</p

Quality control measures of correctly targeted clones.

<p>(A, B) Results from Sanger sequencing. The SNPs rs9940128 (near rs1421085) and rs4783819 (near rs8050136) were amplified and sequenced in the same read to control for possible long deletions in the correctly targeted ESC clones. (C) Two representative karyotypic images. All correctly targeted clones tested displayed a normal karyotype.</p

Schematic representation of the <i>FTO</i> genomic locus (chr16:53,703,963–54,121,941).

<p>(A) SNPs rs1421085 (C/T) and rs8050136 (A/C) are located in the first intron of <i>FTO</i>. (B, C) CRISPR/Cas9 technology was employed to convert ESC line H9 (heterozygous for both SNPs) to homozygosity for both alleles at rs1421085 (C/C or T/T) or rs8050136 (C/C or A/A). Positions of gRNA, PAM sequence and ssODN are indicated by thick lines in blue, purple and black, respectively. SNPs are given in green. Predicted Cas9 cut sites are indicated by red arrow heads.</p

Percentage of HDR, DSB and no cleavage in DMSO-treated and non-treated clones.

<p>Percentage of HDR, DSB and no cleavage in DMSO-treated and non-treated clones.</p