Part:BBa_K4899006

His-tagged dead cas12f, CasMINI

dCasMINI is a modified dcas12f that has been altered by the introduction of three missense mutations in the DNA binding pocket. dCas12f belongs to the class 2 type V-F system family. Members of this family are highly compact nucleases that originate from archaea. dCas12f causes double-stranded breaks by use of its RuvC domain, most versions made by xu et.al (2021) had mutations in this domain preventing cleavage. Additionally, the dCasMINI sgRNA has been modified to significantly reduce its size, while maintaining specificity and binding ability

Usage and Biology

Since their initial discovery by Yoshizumi and colleagues in 1987 (HSU et.al 2014) and the development of the first single guide RNA (sgRNA) by Jennifer Doudna and Emmanuelle Charpentier (JINEK et.al 2012), The CRISPR (clustered regularly interspaced repeats) system and Cas proteins have been applied to a myriad of medical, research, and industrial applications (Hsu et.al 2014, Xu et.al 2021, Zhang et.al2019, Deduna 2020). Cas proteins are RNA-guided DNA endonucleases that cause double-stranded breaks (in most cases). They originate in bacteria and archaea and act as defenses against viruses. Due to the nature of the Cas-RNA complex targeting the complex to the desired location has proven exceptionally easy, with the guide RNA providing the target site any site next to a PAM (protospacer adjacent motif) can be cleaved. The sgRNA can easily be replaced with another similar one targeting another sequence next to a PAM site for cleavage, this means the system is easy to target to a gene of interest (GOI) and very specific. Cas proteins were quickly adopted for research purposes and later wider genetic engineering tasks (Hsu et.al 2014, Xu et.al 2021, Zhang et.al 2019).

Allergenicity testing: Following the methods laid out by the Baltimore Biocrew 2017 team, who discovered that the proteins produced as or by biobricks parts can be evaluated for allergenicity, we decided to test our parts with this system. This was done as the information would be of assistance to any worker/consumer or institution that could desire to use our system for various applications. As well it would also provide an easily available assessment for other igem teams reducing the time needed for literature searches in the start-up phase of their project. In this system the full codon sequence of the part is aligned against databases of known allergens, this happens in full or in overlapping windows of 80 amino acids. For the full sequence alignment the % identity exceeds 50% the biobrick is more likely to cause an allergic reaction For the 80 amino acids sliding window, if the sequence identity is less than 35% the biobrick is less likely to be an allergen (Pearson et.al 1988). CcasMNI with a histag (N terminal) shows a sequence identity of 53% to ragweed homolog of ART1 for a length of 44 amino acids centered on the linker sequence between the tag and the dCasMINI itself. Whilst for the sliding window method no match over 35% was found. as such there is a possibility of the His tagged dCasmini being an allergen, though given the few number of matches, the small length of alignment, and the fact that no matches were found for the sliding window alignment make us believe that it remains unlikely for His tagged dCasMINI is an allergen.

we believe work with this protein can be done in a biosafety level 1 lab.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1417
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1417
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1417
  Illegal BglII site found at 350
  Illegal BglII site found at 359
  Illegal BglII site found at 428
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1417
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1417
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

Optimal conditions for expression 1L flask 500ml culture

Flask: 1L baffled Erlenmeyer flask

Temperature: 16 C

Time from induction: 17 hours

Antibiotics: ampicillin 100ug/mL

Inducer: AHTC 0.05 ug/ml

vector: pASK-IBA2

expression strain: E.coli BL21 (DE3) Gold

promoter: TET

the coding sequence of the part was obtained from IDT as synthesized gBLOCK. Whilst the vector was donated by our supervisors

Characterization

To test if our chosen E.coli BL21 (DE3) Gold bacterial expression system was capable of expressing dcasMINI BL21 (DE3) Gold cells containing the dcasMINI protein in a pASK-IBA2 plasmid were grown on ampicillin agar plates before being picked for liquid cultures. The liquid cultures used LB media and had ampicillin selection. Our initial conditions, 37 oC for 2 hours in a 100 ml non-baffled flask, showed an accumulation of the dcasMINI protein in the non-soluble fraction following lysis (see Results). This was solved by testing expression under a variety of time intervals ( 1,3, and 17 hours) for the time from induction (AHTC to 0.05 ug/mL at OD=0.5) to lysis, and temperatures (16, RT, 20, 30, and 37 o C). It was found that incubation at 16 o C for 16 hours after induction was optimal for the expression of the his-tagged dcasMINI (see figure 1). Under these conditions, there were still large amounts of dcasMINI in the insoluble fraction after cell lysis. Testing was then repeated in larger volumes with 500 ml cultures in 1L flasks. Here we tested using both baffled and nonbaffled flasks as well as time intervals (1 to 17 hours). We found that the optimal time was the same as for the smaller volumes, and baffled flasks produced better results than non-baffled flasks

Figure 1: 4-20% SDS-PAGE gel image of his tagged dCasMINI expression induction overnight at 16 °C. Mechanical lysis with bead beater. Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2 to 5 contain 16 °C overnight induced samples. Results expression, expression testing his tagged dCasMINI expression testing 16 °C+rt, unind: uninduced, ind: induced, insol: insoluble, sol: soluble

For the purpose of purification, we harvested the cells from liquid cultures by centrifugation (6000xg 10 min, rotor = JLA 8.1). The cell pellets were resuspended in binding buffer (recipe at here) before being lysed using a French pressure cell press using 3 runs at 1250 bar (18129 psi). The lysate was then centrifuged at 70000xg for 1 hour to separate the soluble and non-soluble fractions. The 36 mL of cell lysate supernatant was separated from the pellet and filtered through a 2 ul filter. Purification was done by single-step immobilized metal affinity chromatography in a histrap FF 1ml column using an FPLC machine. (buffers and conditions). The histrap FF 1ml column is a NI-NTA column. The sample was loaded on the column at a flow rate of 1ml/min. The column was washed with “buffer A” for 15 CV. Elution was done by a gradient method with a maximum of 400 mM of imidazole in the elution buffer (elution buffers). Figure 2 shows 1 distinct peak in the elution step, this occurred after the elution buffer was applied to the column and is likely to be dcasMINI, a conclusion which is supported by the increase in protein concentration after T9 (see figure 2) as seen in Figure 3 were we see a strong band around 63kDa as expected for dCasMINI. The large UV signal seen in T2 to T6 is the flowthrough of proteins that do not associate with the column. The peak seen in the elution step (early in the gradient, demarked by the spike in the blue line showing the UV signal) has a max detected UV signal of 350 mAU. Purification might be further improved by the use of a stepped gradient strategy, this would give more information about which proteins are eluted more at what concentrations of imidazole. This information would allow for further optimization of the purification process. The UV/VIS data obtained during the purification indicated that there were other proteins that had also bound to the column, shown by the UV curve in Figure 2 samples T13 to T22. This was further supported by SDS page of the produced fractions, showing multiple off band sizes (see figure 3). The fractions with the highest amount of purified protein were chosen to be combined and then the buffer changed. Based on the mentioned UV/VIS data and SDS page shown in figure 3 we decided to use a 30 kDa spin column that helped remove smaller peptides that had been bound to the chromatography column during purification, though the primary purpose remained buffer exchange. This was done by centrifugation in a 20 ml ~30 kDa molecular weight cut-off filtration filter tube (vivaspin centrifugal concentrators) with continuous addition of dilution buffer as the prefilter volume of sample decreased. This was done to better facilitate long-term storage.

Figure 2: Chromatogram of his tagged dCasMINI purification with a HisTag FF column results for his tagged dCasMINI, using HisTrap FF, 1 ml column. conducted with ÄKTA start protein purification system.

Figure 3: 4-20% SDS-PAGE gel of fractions collected from his-tag purification of his tagged dCasMINI construct. The first lane contains the PageRuler™ Plus Prestained protein Ladder 10-250. Lanes 2 and 3 contain samples collected before the culture was pelleted and samples were taken from the supernatant during loading on the affinity column after dilution. The annotation in the figure of F# indicates which fraction the lane contains a sample from. Of the fractions most of the proteins in the sample are in F3. However, the largest amount of purified protein is present in lanes F11 to F15, with most of the purified sample being in F11.

To show that dcasMINI binds its guide RNA, the isolated dcasMINI was incubated with one of the sgRNAs in an RNA-protein binding buffer (buffers) for 15 minutes at 4 degrees. The complex was then mixed with a native gel loading buffer before being run in a native gel (PAGE 4-16% Bis-Tris). Initial attempts for staining showed no RNA or protein (staining solution with a concentration of 3x gelred for 60 min). Therefore other methods were attempted (silver staining). The resulting EMSA native-PAGE gel showed no bands for the protein in the concentrations used in the reaction mix, appropriate band sizes were present in control serial dilution. In this gel (see figure 4) we could see bands as expected for dCasMINI at higher concentrations. The incubation reaction was also run on a 2% agarose gel where the RNA bands were apparent, though no band above the expected size of the sgRNA (184 bp) was present, which would have indicated binding of the dcasMINI protein. We concluded then that the current conditions do not easily facilitate binding

Future work Further characterization of part can be best achieved by optimizing the binding conditions for the guide RNA to the dcasMINI protein. As the in vitro binding conditions for dcasMINI with its re-engineered guide RNA have not been characterized, multiple conditions should be tried for EMNSA to find the optimal buffer. As well as the use of an FPLC or HPLC machine for size-exclusion chromatography with a standard gel filtration or analytical column. This would provide data that better characterize the binding of the guide RNA and dcasMINI, as well as more definitive proof that would be provided by the UV absorption spectra.

Referanses

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-1278. doi: 10.1016/j.cell.2014.05.010. PMID: 24906146; PMCID: PMC4343198.

Martin Jinek et al.,A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity.Science337,816-821(2012).DOI:10.1126/science.1225829

Xu, X., Chemparathy, A., Zeng, L., Kempton, H. R., Shang, S., Nakamura, M., & Qi, L. S. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Molecular Cell, 81(20), 4333-4345.e4. https://doi.org/10.1016/J.MOLCEL.2021.08.008

Doudna, J.A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020). https://doi.org/10.1038/s41586-020-1978-5

W.R. Pearson & D.J. Lipman PNAS (1988) 85:2444-2448