TAG-IN Design

About

Tool Structure and Scoring

TAG-IN utilises a number of existing scoring models and tools:

  1. "Rule Set 2" scoring model by Doench et al. 2016 to assess sgRNA efficiency.
  2. Off-target searching up to 3 mismatches for both NGG and NAG PAM using Bowtie Langmead et al. 2009.
  3. Off-target scoring model by Hsu et al. 2013 to assess sgRNA specificity.
  4. And a novel ssDNA design function which allows facile single strand DNA (ssDNA) design for 3 prime tagging.
  5. Visualisation of genomic data was facilitated by graphics library Scribl.

ssDNA Design

ssDNA design is completed with the following steps:

  1. Retrieve gene annotation from Ensembl database.
  2. Extract restrained sequence around annotated stop codon.
  3. For each sgRNA simulate sgRNA::Cas9 complex cleavage 3 nt upstream of PAM sequence.
  4. Tag of choice inserted as close to the cleavage site as possible.
  5. Resulting PAM 200nt ssDNA sequence contains tag of choice inserted immediately adjacent to the stop codon with flanking arms homologous to target sequence.
  6. PAM sequence is mutated to prevent further Cas cleavage of donor sequence.

Summarisation score

In order to speed up user selection a heuristic summary score was created:



where S represents the summarisation score for sgRNA within the intron or 3UTR, d the distance from the stop codon, m the MIT off-target score by Hsu et al. 2013, and r represents the RS2 efficiency score by Doench et al. 2016. To minimise potential for CRISPR activity within the coding sequence sgRNA that act within the intron/exon are penalised and hence governed by equation (2). Minimising sgRNA distance, d, from the stop codon was considered important to maximise efficiency of tagging - with a particular preference between 8-15 bp from the stop codon.



Please contact us with any queries or concerns:

Pollard Lab - Email: s1578687@sms.ed.ac.uk