CRISPR Toolbox for Genome Regulation
We have been developing a kit of CRISPR tools, repurposed from various bacterial CRISPR/Cas9 systems. The toolkit allows sequence-specific regulation of gene expression on the transcriptional or epigenetic levels, as well as for genome imaging in living cells. Please click on each topic to expand for more details.
We have engineered the bacterial immune CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system as a platform for RNA-guided gene in bacteria or human cells. This CRISPR interfering (CRISPRi) method, works independently of host cellular machineries, requiring only a nuclease-deactivated Cas9 (dCas9) protein and a customized single guide (sg) RNA designed with a 20-basepair complementary region to any gene of interest. Co-expression of dCas9 and sgRNA can efficiently block transcription (in bacteria, ~ 300-fold repression) by interfering with transcriptional elongation, RNA polymerase binding, or transcription factor binding.
The binding specificity is determined jointly by a 20-bp matching region on the sgRNA and a short DNA motif (protospacer adjacent motif or PAM, sequence: NRG, R = G or A) juxtaposed to the DNA complementary region. The uniqueness of CRISPRi, as compared to several recently published works on using the wild-type CRISPR system for genome editing, is that the nuclease-deficient dCas9 mutant of could silence transcription of the target the gene expression without genetically altering the target sequence. Thus, CRISPRi is a system that can regulate a genome instead of modifying a genome.
More details about the CRISPRi technology can be found on wikipedia.
CRISPRi/a Computational Tool
We have created a designer tool for genome editing, repression and activation, named CRISPR-ERA (E = editing, R = repression, A = activation). The tool allows generation of sgRNAs for gene activation or repression using our pre-assmebled databases of using CRISPR for transcriptional repression or activation in different organisms. Currently, nine organisms including two bacterial speices E.coli, B. subtilis; one yeast: S. cerevasiae; C. elegans, fruit fly, zebrafish, mouse, rat, and human databases are included. It also enables automated genome-wide sgRNA design.