CRISPR interference genome engineeting toolbox
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.
1. CRISPRi transcription regulation system in bacteria (E. coli) | Material Requests
The first plasmid (pdCas9_bacteria) contains an anhydrotetracycline (aTc)-inducible dcas9 gene on a p15A vector with chloramphenicol resistance. The second plasmid (pgRNA_bacteria) contains a constitutive sgRNA expression cassette on a ColE1 vector with ampicillin resistance, wherein N20 can be custom designed to target arbitrary sequences in the genome. Co-expression of both plasmids in bacteria could cause about 300-fold repression on targeted genes.
2. CRISPR-mediated transcription regulation system in eukaryotes | Material requests
CRISPR transcription regulation system in human cells
The dCas9 fusion plasmids contain a human codon optimized dcas9 gene that is fused to different effector domains, under the control of either a spleen focus-forming virus (SFFV) or a murine Stem cell retrovirus LTR promoter. The sgRNA plasmids contain a murine U6 promoter controlled sgRNA cassette, wherein the GN19 can be custom designed to target sequences in the genome. The sgRNA plasmid also contains a CMV-puro-t2A-mCherry expression cassette, for selection or fluorescent gating of transiently transfected cells.
CRISPR transcription regulation system in yeast
The dCas9 fusion CEN/ARS plasmids contain a human codon optimized dCas9 fused with two C-terminal SV40 nuclear localization signal sequences and an Mxi1 repressive domain. The sgRNA CEN/ARS plasmids contain a SNR52 promoter, sgRNA, and SUP4 terminator 3’ flanking sequence