Difference between revisions of "Part:BBa K2316000"

Revision as of 12:46, 1 November 2017

∆RuvCIII-2 ∆HNH ∆REC2 Sp-dCas9

dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner. This is an improvement from part BBa_K2130000

In our attempt to improve our truncations, we explored 2 possible avenues of improvements. First, we considered if further truncations is possible, by looking at additional combinations and novel truncations. Second, we considered alternative functionalities for our truncated dCas9.

Based on our attempts at further combinations of truncations as well as truncation of newly characterized domains, we noted that ∆3ple, a combination of ∆HNH ∆REC2 and ∆RuvCIII-2, is the best combination of truncations we have. We noted that transcription activation functions of our ∆3ple is poor. Thus, we are interested if ∆3ple is functional in other applications, where poorer target DNA affinity may be less important. We decided to test the CRISPRi functionality of 3ple. As a proof of concept, we designed the experiment for test in bacteria.

In bacterial CRISPRi, dCas9 is targeted to -35 or-10 of promoter to block RNA polymerase binding, or to the non-template (NT) strand of the gene to block RNA polymerase transcription from proceeding. Transcription is blocked, resulting in expression interference.

For our construct, we decided to carry out CRISPRi on an endogenous GFP reporter E coli strain (a kind gift from Swaine Chen lab). BPK 65767 is adapted to non-T7 expression. The Cas9 promoter is replaced with Lac inducible promoter(Part BBa_K314103) while the sgRNA promoter was replaced with high constitutive promoter(Part BBa_J23100). T7 terminators were maintained, as it has been shown that T7te can terminate normal promoters.

We tested targeting of -35, -10 and NT, using the original bacterial optimized Cas9 in BPK 65757 mutated at D10A and H849A to make dCas9. While -35 inteference leads to partial repression of GFP expression, -10 and NT inteference leads to robust CRISPRi of GFP. 2 negative controls were used, one with scrambled gRNA target, another with a target not available in the GFP reporter E coli strain. Both negative control confirmed no CRISPRi function against GFP gene with IPTG induction.

Thus, -10 target is chosen for our tests with truncated dCas9. WT-dCas9 and truncated dCas9 variants were cloned into BPK 65767, replacing Cas9 – to account for human optimized codons. The CRISPRi experiment is then repeated. Cells are immediately induced at OD0.2 with 0.5mM IPTG. Fluorescence (485nm/516nm) is normalized to cell density (OD600). At T=4hours, CRISPRi is observed for all samples. In both T=4 and T=12, induction uniformly led to robust CRISPRi of GFP expression for all truncated dCas9 variants – including ∆3ple.

Results suggest that multiple truncated ∆3ple can still be used to great effect for CRISPRi applications, at least in the context of bacterial genomes. The corresponding decrease in activity with increasing truncations seen in VPR fusion gene activation was not observed in CRISPRi. The poorer target DNA binding affinity of our multiply truncated dCas9 may be less important in CRISPRi, compared to in transcription activation, where considerably longer dwell time could be required for effective VPR fusion activity.

Usage and Biology

For this part, we have deleted the HNH domain, Rec2 domain and the RuvCIII-2 region of the SP-dCas9 and tested it's binding capability by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator.

Sequence and Features