Cas9-based kill switch

Mutagenesis PCR

To make mutations in dCas9, mutagenesis PCR was used according to the Quikchange protocol. The adapted protocol that worked best for us is shown here.
Primers with mismatches to the template on the site of the mutation were designed using their online tool.

The following PCR mixture was made:

Yeast assembly

To construct pEVOL-Asp, a construct containing an aminoacyl synthetase (aaRS) and tRNA_CUA (ref) suitable for incorporating Aspartate in response to the TAG stopcodon, yeast assembly was used. In this method, linear DNA fragments are transformed into Saccharomyces cerevisiae. To select for positive clones, one of the fragments is a part of the yeast expression vector pYES2. pYES2 contains the URA3 gene, which releases auxotrophy for uracil, as well as a 2µ ori for high copy maintenance in yeast. The other fragments included two IDT gBlocks containing the aaRS and tRNA_CUA, as well as part of pEVOL-pAzF(ref) containing the arabinose promoter and CAT resistance gene.

Primers to generate fragments for yeast assembly were made using the href=”http://nebuilder.neb.com/”>NEBuilder for Gibson assembly, with 40 bp overlap between fragments. PCR was performed as usual (hyperlink), fragments were checked by gel electrophoresis and PCR products were purified using the PCR cleanup kit from Zymo.

Homemade heat-shock competent S. cerevisiae cells were incubated at RT for 10 minutes prior to mixing with ~400 ng of each PCR product and 100 µg sheared salmon sperm DNA (Sigma Aldrich). 700 µL of 1x LiAc/ 40% PEG-3350/ 1x TE was mixed in and solutions were incubated at 30°C for 30 minutes. Next, 88 µL DMSO was added, samples were mixed and heat shocked at 42°C for 7 minutes. Cells were pelleted in a microcentrifuge, washed with 1 ml 1x TE, resuspended in 100 µL 1x TE and plated on YPD plates. Plates were incubated over the weekend at 30°C.

To isolate the DNA from possible correct clones, a modified miniprep protocol was followed.
Colonies were inoculated in 1.5 ml YPD medium and grown overnight while shaking at 30°C. Cells were harvested in a microcentrifuge (13000 rpm, 5 minutes) and the pellet was resuspended in 100 µL STET buffer. To lyse the cells, they were disrupted in a bead beater, another 100 µL STET was added and samples were boiled for 3 minutes in a heat block. Samples were spinned down again in a microcentrifuge, and the supernatants were used to proceed with miniprep as usual (hyperlink). This gave fairly low plasmid yields (~30 ng/ µL), but enough to do PCR and transform into E. coli.

Correct construction of the plasmid was confirmed by PCR and sequencing.

YNB-UDM medium/plates

Cloning of gRNA plasmids using annealed oligos

Cloning was performed in the same way as normal restriction-ligation cloning (hyperlink), but annealed oligonucleotides were used as insert.

Oligos were annealed as in Jao et al(ref), but using 1x ligase buffer instead of NEBuffer 3.
Two complementary oligos corresponding to the target sequence were diluted to 10 µM each in T4 ligase buffer (NEB) to a final volume of 20 µL. To anneal them, the following protocol was used:

5 min denaturation at 95ºC, cooling to 50ºC at -0.1ºC per second, remaining at 50ºC for 10 minutes, followed by cooling to 4ºC at -1ºC per second.

The annealed oligos were cloned into pT7-gRNA (Addgene plasmid # 46759). pT7-gRNA was digested with BsmBI and BglII for 1 hour. Cut plasmids were checked by gel electrophoresis, and digested bands were purified from the gel (hyperlink).

Next, we proceeded with ligation as usual (hyperlink), diluting the annealed oligos 16x to add 1 µL to 25 ng digested pT7-gRNA. Ligation mixtures were diluted 2x, and 1 µL was used for electroporation (hyperlink).

in vitro RNA transcription and purification

Note: always use gloves and filtered tips when working with RNA, as RNAses can contaminate your samples

in vitro transcription

Prior to transcriptions, pT7-gRNA plasmids were digested with BamHI and purified. RNA guides were transcribed in vitro using the HiScribe high yield RNA synthesis kit from NEB. Reactions were assembled using the following protocol:

Cas9 expression and FPLC purification

Day 1
Cas9 was expressed in a recoded E. coli strain lacking TAG stopcodons and release factor 1(C321dA; ref), but higher expression can probably be achieved using a bacterial strain more suited to protein expression.

10 ml overnight cultures were prepared of C321dA transformed with Cas9-expresso, dCas9-expresso, Ala10TAG-expresso + pEVOL, or Ala840TAG-expresso + pEVOL. Cultures were grown in LB in the presence of 35 µg/mL kanamycin (expresso constructs) and 25 µg/mL chloramphenicol (pEVOL constructs) when appropriate.

Day 2
Overnight cultures were diluted 100x in 0.5 L LB, with appropriate antibiotics, and divided over two 1L erlenmeyers. Cultures were were grown to an OD600 of about 0.8 at 37ºC while shaking. Next, cultures were put at 20ºC shaking for 30 minutes (cold shock) to induce expression of chaperones that aid in protein stability.
Cas9 expression was induced by adding rhamnose to 0.2%. In cultures that contained a pEVOL vector, expression of the aminoacyl synthetase was induced by adding arabinose to 0.2%. When appropriate, at this time also the synthetic amino acid was added to a concentration ranging from 200 µM to 1 mM. Cultures were shaken overnight at 20ºC.

Day 3
All centrifugation steps were performed at 4 ºC, and samples were kept on ice.
Cells were harvested by centrifugation at 4000 x g for 15 minutes. Supernatant was removed, and cells were washed once in 50 ml Tris-HCL buffer, pH7. After spinning down the cells again, they were resuspended in 10 ml cold His buffer A. Cells were lysed using a chilled French press cell (pressure ~30,000 psi) and lysed samples were spinned at 16000 x g for 10 minutes. Supernatants were collected, filtered (0.45 µm filter) and purified using FPLC.

Since C321dA is not optimized for protein expression, we aimed to reduce protein breakdown by adding cOmplete protease inhibitor (hyperlink) to our samples before lysis. However, high yield of Cas9 was also observed when this protease inhibitor was omitted.

Using the expresso vector for Cas9 expression introduced a C-terminal his-tag to our Cas9 constructs, which was used for protein purification. An AKTA Purifier system was used, with a 1 mL HisTrap HP column (GE Healthcare Life Sciences) that was equilibrated with His buffer A . The cell-free extracts were loaded on the column, and the column was washed by addition of 2% His buffer B to already remove some unspecifically bound proteins. Elution was performed with a gradient of His buffer B.

His buffer A

in vitro Cas9 cleavage assay

Cleavage assays were performed according to the href=”https://www.neb.com/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386”>NEB protocol, but with our own Cas9 samples and homemade 5x Cas9 assay buffer.

The following reaction mixtures were assembled: