Overview
To increase the accuracy and specificity of the detection, we developed an assay over our Paired dCas9 Reporter (PC Reporter) System to get more sequence information from the target genome. The core as well as the first step of the design of the array is to screen over the entire genome and get paired specific sequences (CRISPR target sites) with high specificity as markers.
We develop a method named SSPD to achieve our aim, which is composed of 4 steps:
- Search for guide sequences of gRNA candidates
- Specificity test for each candidate
- Pair left and right target sites with optimal spacer length
- Design PCR fragments
SSPD Methods
Search for guide sequences of gRNA candidates
Recall the structure of Paired dCas9 Reporter (PC Reporter) System, a protospacer adjacent motif (PAM) sequence in the form of 5’-NGG-3’ at 3’ end of guide sequence, usually 20bp, on the non-complementary strand. (Figure 1) As it was showed in our experimental results that PAM-out orientation (5’-CCNN20-…-N20NGG-3’) was highly efficient for PC Reporter system to work, thus our model would focus on this orientation. (However, it can be more convenient to adjust our program for guide sequence design also with other orientations. See more in Supplementary 1 )
Figure 1. Schematic illustration of guide design in PAM-out orientation. Note the 20nt guide sequence is identical to target non-complementary strand.
We took advantage of Python 3.4.3 build-in regular expression to search for left guide sequences of gRNA (‘(?<=cc).(?=.{20})’) and right guide sequences of gRNA (‘(?<=.{20}).(?=gg)’) separately, which would be paired later for PC reporter system to function.
Oligo Generator
Using SSPD method mentioned above, we can easily find the reliable target sites on genome. However, designing multiple target sites into oligonucleotides sequences for following sgRNA construction manually can be laborious. Thus here we developed a supplementary program to facilitate oligo sequence generation, which is combined with our sgRNA generator (Link to Part xxx, Figure 6). Specifically, we used Golden Gate Cloning to make it more convenient to substitute guide sequences for different target sites. Detailed operation is explained on flow chart below. See more details in Supplementary 2.
Figure 7. Schematic illustration of a flow chart explaining the protocol of guide sequence substitution.
