Part:BBa_K4899004

dCasMINI, a catalytically inactive Cas12f

a version of Cas12f that has 3 mutations in the DNA binding pocket to reduce of target effects and a mutation in the RuvC domain to render it catalytically inactive. useful for targeting other domains

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.al 2019, 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.al2019).

To help expand the possible applications of CRISPR systems Xu et.al (2021). set out to engineer a “miniature CRISPR-Cas system for mammalian genome regulation and editing”. Their goal was to generate a compact, efficient, and specific system for mammalian genome engineering. The result of this was casMINI. CasMINI is a modified cas12f that has been altered by the introduction of three missense mutations in the DNA binding pocket. Cas12f belongs to the class 2 type V-F system family. Members of this family are highly compact nucleases that originate from archaea. Cas12f 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 CasMINI guide RNA has been modified to significantly reduce its size, while maintaining specificity and binding ability. Cas12f was chosen by the authors due to its is a naturally small Cas effector of 400-700 amino acids in size compared to the more commonly used Cas9 and Cas12a with sizes of 1000 and 1500 amino acids respectively(xu et.al 2021). The researchers took this into account as they reasoned that a reduction in size could help reduce the problem of applying these Cas proteins in other organisms. Previous medical and genome engineering applications of cas proteins have proven challenging, as their protein and coding sequence sizes have made them difficult to load into vectors, like lentiviruses, adeno-associated viruses, and lipid nanoparticles as both mRNA and proteins (Wang et.al 2020, Xu et.al 2021). This has hampered their effective use in various medical and research applications, as more complex cas fusion proteins have been largely limited in use to settings where large DNA/RNA molecules or proteins can be directly transfected into the organism (Xu et.al 2021, Wang et.al 2020). This complicates their use on most multicellular organisms after early embryo development.

The primary purpose of the generated CasMINI construct was to specifically target functional domains used in genome engineering and epigenetic research. For this reason, the introduction of double-stranded breaks at the target would be highly disruptive to the research question at hand. For this application, the researcher generated several catalytically inactive (dead) Cas12f that do not have the ability to induce double-stranded breaks, annotated dCasMINI.

dCasMINI and CasMINI have a series of DNA binding pocket mutations introduced by Xi et.al (2021), D326A/D510A/D143R/T147R/K330R/E528R in the authors data this seems to have increased either the specificity or strength of binding reducing off target effect.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1354
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1354
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1354
  Illegal BglII site found at 287
  Illegal BglII site found at 296
  Illegal BglII site found at 365
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1354
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1354
  
• 1000
  COMPATIBLE WITH RFC[1000]

allergenicity

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, if the sequence identity exceeds 50% the biobrick is more likely to cause an allergic reaction For the 80 amino acids sliding window, a sequence identity of less than 35% the biobrick is less likely to be an allergen (Person et.al 1988. For dCasMINI we find no matches for either the full sequence alignment or the 80 mer sliding window alignment. This indicates that dCasMINI is unlikely to cause an allergic reaction.

Collection and Variants

The UiOslo iGEM team did not characterize the pure dcasMINI protein, rather it was used to create two composite parts. These two composite parts His tagged dCasMINI and Strep tagged dCasMINI were combined with the corresponding tag. These protein variants may have different properties, but as the changes are not large the characterization should be partly correct for the dcasMINI as well. Both of the tagged versions have been characterized and the information is uploaded to the corresponding parts registry. Furthermore the dcasMINI may be combined with the two sgRNAs that were designed specifically for it, these can be found at sgRNA1 and sgRNA2

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

Wang, Dan, Feng Zhang, and Guangping Gao. "CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors." Cell 181, no. 1 (2020): 136-150. Accessed October 12, 2023. https://doi.org/10.1016/j.cell.2020.03.023.

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