Coding

Part:BBa_K3636000

Designed by: Daria Smolyarova, Nadezhda Korostyleva, Anastasia Kislova   Group: iGEM20_Moscow   (2020-10-21)


CasX (Cas12e)

Usage and Biology

What is CasX?

The CRISPR/Cas system initially serves as an adaptive immunity against invading nucleic acids in prokaryotes. On the other hand, this system is widely used as a tool for nucleic acids detection and genome editing, for which The Nobel Prize in Chemistry 2020 to Emmanuelle Charpentier and Jennifer A. Doudna was awarded[1]. CasX, also well-known as Cas12e [2] is an enzyme related to another RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a. CasX was discovered by metagenomic analysis of microbial DNA from groundwater samples. The wild-type CasX expressed by Deltaproteobacteria, a class of Proteobacteria[3][4]. CasX differs from other Cas enzymes in sequence and biochemical properties, therefore, CasX became a third full-fledged unique platform for RNA-programmed genome editing[5].

The place of CasX in Cas classification

The CRISPR-Cas systems are divided into two classes: сlass 1 includes types I, III and IV, class 2 includes types II, V and VI (Table 1). The types are divided into subtypes (II-A, II-B, and II-C for type II)[6]. Сlass 1 Cas proteins perform their effector functions with help of multi-protein complex, while the class 2 have one multi-domain protein. Effector proteins of Cas9 and Cas12 have a domain with endonuclease from the RuvC family that initiates cleavage of the DNA strand not complementary to the guide RNA (nickase activity)[7][8]. According to phylogenetic analysis CasX originated from a TnpB-type transposase by an independent insertion event into ancestral CRISPR loci, distinct from Cas12a and the remaining type V effectors[9].

Table 1. Simplified classification of some Cas systems T--Moscow--Table1full.png

A structure of CasX

CasX contains both domains analogous to other Cas proteins: Helical-I, Helical-II, OBD (oligo binding domain, RuvC and a BH (bridge helix) as well as completely novel: NTSB (non-target strand binding) and target-strand loading (TSL) (Fig. 1).

NTSB domain contains a four-stranded beta-sheet and sits next to the non-target strand of the DNA. TSL domain is located in a position analogous to that of the so-called “Nuc” domain of other enzymes of type V CRISPR-Cas systems. TSL domain is responsible for target strand position in the RuvC active site. In the AsCas12a “Nuc” domain, amino acids Arg1226 and Asp1235 promotes target strand cleavage. In the CasX “Nuc”-analogous domain, the TSL, residues Arg917 and Gln920 interact with DNA (NTS in State I and TS in State II) that is adjacent to the active site. The NTSB domain is used for DNA unwinding[10]. There is a point of view that the NTSB and TSL of CasX aid in ‘proofreading’ base pairing throughout the protospacer for cleavage rather than aiding in binding[11].

T--Moscow--Cas12eSmall.png
Figure 1. A structure of CasX PDB:6NY2

In the CasX-sgRNA binary complex the guide RNA is ~26% of the mass according to complex structure. It is much more than can be observed in other CRISPR-Cas effector complexes type II or V (~8% in LbCas12a, ~20% in AacCas12b, and ~16% in SpyCas9)[12], because the CasX is much smaller than Cas12 or Cas9 [13](Table 2). Size matters because it is easier to work with smaller proteins and they can fit in adeno-associated virus (AAV) while vector delivery.

Table 2. Approximate size of different Cas proteins
T--Moscow--Table2.png

The properties of CasX

Both Cas9 and Cas12 (including CasX) systems create double strand breaks and identify the target by protospacer adjacent motifs (PAMs). But the process of binding and cleavage of DNA by Cas9 and Cas12 are different: Cas9 recognizes a 3′-G-rich PAM and produces blunt ends cleaved by the RuvC and HNH domains, whereas Cas12 recognizes a 5′-T-rich PAM and produces sticky ends cleaved solely by the RuvC domain[14]. CasX generates products with ~10-nucleotide sticky ends due to cleavage 12–14 nucleotides after the PAM on the non-target strand and 22–25 nucleotides after the PAM on the target strand. Cas X also has a trans-ssDNA cutting activity, but it is minimal in comparison with Cas12. Mutations in the PAM or mismatches within the protospacer completely prevent cleavage. Mutations outside the PAM and protospacer affect affinity. Specificity within the PAM is stronger than for SpCas9 and AsCas12a, but is comparable in binding energy to SpCas9:Nanog in the protospacer[15][16].

CasX is capable of genome silencing and editing. That was shown for bacterial and eukaryotic cells[17]. Also CasX has nikase activity, what allows it to be used in detection systems like SHERLOCK[18] and DETECTOR[19].

Design Notes

We conducted a bioinformatical analysis that showed that there are four restriction sites in the CasX sequence that are incompatible with the Assembly Standards. In future work, we will need to include four silent point mutations to exclude those restrictions sites. We designed eight primers that will allow us to conduct this work.

Primers for 4 point mutations:

  1. noEcoR1-F-58
    acgaaaaggaattTtatgcctgtgaga
  2. noEcoR1-R-58
    tctcacaggcataAaattccttttcgt
  3. noSpe1-F-59
    ttacgaggggcttacAagtaaaacgtattt
  4. noSpe1-R-59
    aaatacgttttactTgtaagcccctcgtaa
  5. no1Ng04-F-60
    gaagaagccCgccaaacgt
  6. no1Ng04-R-60
    acgtttggcGggcttcttc
  7. no2Ng04-F-59
    gatgagccCgccctgttt
  8. no2Ng04-R-59
    aaacagggcGggctcatc

Source

Addgene plasmid # 126418 ; http://n2t.net/addgene:126418 ; RRID:Addgene_126418


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. ↑ 1 Press release: The Nobel Prize in Chemistry 2020 (https://www.nobelprize.org/prizes/chemistry/2020/press-release/)
  2. ↑ Eugene V. Koonin, Kira S. Makarova, and Feng Zhang, “Diversity, Classification and Evolution of CRISPR-Cas Systems,” Current Opinion in Microbiology 37 (June 2017): 67–78, https://doi.org/10.1016/j.mib.2017.05.008.,
  3. ↑ Jun-Jie Liu et al., “CasX Enzymes Comprise a Distinct Family of RNA-Guided Genome Editors,” Nature 566, no. 7743 (February 2019): 218–23, https://doi.org/10.1038/s41586-019-0908-x.
  4. ↑ David Burstein et al., “New CRISPR-Cas Systems from Uncultivated Microbes,” Nature 542, no. 7640 (February 9, 2017): 237–41, https://doi.org/10.1038/nature21059.
  5. ↑ Liu et al., “CasX Enzymes Comprise a Distinct Family of RNA-Guided Genome Editors.”
  6. ↑ Eugene V. Koonin and Kira S. Makarova, “Origins and Evolution of CRISPR-Cas Systems,” Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772 (May 13, 2019): 20180087, https://doi.org/10.1098/rstb.2018.0087.
  7. ↑ Hiroshi Nishimasu et al., “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA,” Cell 156, no. 5 (February 2014): 935–49, https://doi.org/10.1016/j.cell.2014.02.001.
  8. ↑ Jun-Jie Liu et al., “CRISPR-CasX Is an RNA-Dominated Enzyme Active for Human Genome Editing,” Nature 566, no. 7743 (February 2019): 218–23, https://doi.org/10.1038/s41586-019-0908-x.
  9. ↑ Liu et al., “CasX Enzymes Comprise a Distinct Family of RNA-Guided Genome Editors.”
  10. ↑ Liu et al., “CRISPR-CasX Is an RNA-Dominated Enzyme Active for Human Genome Editing.”
  11. ↑ Liyang Zhang et al., “Systematic in Vitro Profiling of Off-Target Affinity, Cleavage and Efficiency for CRISPR Enzymes,” Nucleic Acids Research 48, no. 9 (May 21, 2020): 5037–53, https://doi.org/10.1093/nar/gkaa231.
  12. ↑ Alexis C. Komor, Ahmed H. Badran, and David R. Liu, “CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes,” Cell 168, no. 1–2 (January 12, 2017): 20–36, https://doi.org/10.1016/j.cell.2016.10.044.
  13. ↑ Hui Yang and Dinshaw J. Patel, “CasX: A New and Small CRISPR Gene-Editing Protein,” Cell Research 29, no. 5 (May 2019): 345–46, https://doi.org/10.1038/s41422-019-0165-4.
  14. ↑ Liu et al., “CRISPR-CasX Is an RNA-Dominated Enzyme Active for Human Genome Editing.”
  15. ↑ Yang, H., Patel, D.J. CasX: a new and small CRISPR gene-editing protein. Cell Res 29, 345–346 (2019). https://doi.org/10.1038/s41422-019-0165-4
  16. ↑ Zhang et al., “Systematic in Vitro Profiling of Off-Target Affinity, Cleavage and Efficiency for CRISPR Enzymes.”
  17. ↑ Liu et al., “CRISPR-CasX Is an RNA-Dominated Enzyme Active for Human Genome Editing.”
  18. ↑ Max J. Kellner et al., “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases,” Nature Protocols 14, no. 10 (October 2019): 2986–3012, https://doi.org/10.1038/s41596-019-0210-2.
  19. ↑ James P. Broughton et al., “CRISPR–Cas12-Based Detection of SARS-CoV-2,” Nature Biotechnology 38, no. 7 (July 2020): 870–74, https://doi.org/10.1038/s41587-020-0513-4.
[edit]
Categories
Parameters
None