CRISPRi/a Toolbox for Genome Regulation
We have developed a toolkit of CRISPRi/a tools from various species of bacteria CRISPR systems. The toolkit allows efficient sequence-specific activation or repression of gene expression in different organisms (bacteria, yeast, mammalian cells). We also developed the CRISPR for tracking chromatin dynamics in living mammalian cells. Please click on each topic 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, yeast, and 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.