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
To increase the accuracy and specificity of the detection, we develop an assay over our Paired dCas9 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 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 candidates
Specificity test for each candidate
Pair left and right guides with optimal spacer length
Design PCR fragments
We will introduce each step in detail separately with analysis about Mycobacterium tuberculosis (TB) genome as an example. After the target sites are chosen, we developed an Oligo Generator to turn the target sites into oligonucleotides sequences for following sgRNA construction combined with our gRNA generator.
SSPD Methods
Search for guide 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 is 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)
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 and right guide sequences of gRNA separately, which would be paired later for PC reporter system to function.
Instructors
OUYANG Qi
LOU Chunbo
