TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>

[GUIDELINES]
<div title="Page 7">
<div>
<div>
<p>Mutations and off-target effects<br /> The method has various safeguards against the effects of mutations and off-target cutting. Most importantly, unlike in many uses of CRISPR to create DSBs, the rate of indel formation at the DSB site should be low, as LOH occurs through high fidelity HR, rather than error-prone non-homologous end joining. In the LOH process, the targeted cut site is gene-converted to the PAM-less allele, abolishing further cutting by Cas9. Off-target LOH events are uncovered in our whole-genome genotyping. Finally, in genetic mapping using LOH events a positive signal would typically require multiple different recombination events to generate similar phenotypes, so gRNA-specific off-target effects or mutations should not produce false positive signals.</p>
</div>
</div>
</div>
<div title="Page 8">
<p>We checked whether the LOH process introduced mutations at the targeted cut site by Sanger sequencing in 72 independent LOH lines, and did not observe any mutations. We amplified genomic DNA in an interval surrounding the cut sites of 78 individuals, sampled from both the Chr 7L panel and the manganese fine-mapping panel. The PCR products were Sanger sequenced from both ends; we obtained high quality sequence data from both reads for 72 of the 78 individuals. The resulting data were analyzed using the R package SangerseqR (34) to make homozygous as well as heterozygous base calls. Heterozygous calls were made with a cutoff of 0.25 for the ratio of intensity for the secondary basecall relative to the intensity of the primary basecall (default is 0.33). Mutation calls were made if a position had an observed mutation in both of the paired sequencing reads.</p>
<p> </p>
<div title="Page 8">
<div>
<div>
<p>Power calculations<br /> We investigated the relationship between statistical power, effect size, and proximity of a QTL to the telomere, in the panel of 384 LOH strains with LOH targeted uniformly across Chr 7L. With this panel design we expect power to detect a QTL to be highest near the telomere and lower for QTL near the centromere. Smaller fractions of lines will be homozygous for polymorphic positions near the centromere than for polymorphic positions near the telomere. We simulated a single additive QTL at each variant site on Chr 7L using our observed genotype data, by generating a simulated phenotype for each line drawn from a normal distribution with variance 1 and mean 0, β, or 2β, depending on the line’s genotype at the variant (homozygous BY, heterozygous BY/RM, or homozygous RM). Simulated phenotypes were generated for the 384 LOH lines while varying the simulated QTL’s β from 0.25 to 5, at each of 2000 BY/RM polymorphic positions along Chr 7L, 100 times. Heritability (h2) was calculated as the ratio of var(g) to var(p). QTL mapping was performed as described above. Power was calculated at each QTL position and effect size as the fraction of simulations out of 100 with a LOD greater than 3 at the simulated QTL position.</p>
</div>
</div>
</div>
<div title="Page 9">
<p> </p>
<p>Data deposition<br /> Code and data can be found at https://github.com/joshsbloom/crispr_loh; sequencing data is deposited at the Sequence Read Archive under accession SRP072527.</p>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.