Coding

Part:BBa_K2965021

Designed by: Wenxiao Li   Group: iGEM19_ZJU-China   (2019-10-15)
Revision as of 03:46, 28 October 2020 by Fangzhiyu (Talk | contribs) (→‎Improvement)

AsCas12a (AsCpf1)

AsCas12a (AsCpf1) comes from Acidaminococcus sp. BV3L6, it is a programmable DNA endonuclease guided by a single CRISPR RNA (crRNA). Targeting requires a crRNA complementary to the target site as well as a 5' TTTN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Once it recognizes its target, it will be activated and exhibits a “collateral effects” of promiscuous DNase activity.

Usage and Biology

AsCas12a RNPs bind to DNA and start to move along it by a hopping mechanism. When the AsCas12a RNPs encounter a potential target site containing a PAM sequence, they begin to initiate R-loop formation by forming base-pair hybrids between the crRNA and the target DNA strand. If sufficient matched nucleotides exist in the PAM-proximal region, the R-loop formation will be stable. After the stable R-loop conformation is formed, AsCas12a, via the activity of its RuvC domain, first cleaves the non-target DNA strand and then cleaves the opposite, target DNA strand, regardless of the presence of a PAM sequence. Finally, AsCas12a rapidly releases the PAM-distal DNA fragment but continues to hold the PAM-proximal portion of DNA[1].


Characterization

Results

We link AsCas12a fragment and pET-28a(+) plasmid to form AbCas12a expression plasmid with His tag. Then we transfer the recombinant plasmid into E. coli BL21 and test it by colony PCR. Result is shown in Figure 1 below. We also verify it by TSINGKE Biological Technology Institute sequencing.

Figure 1. The result of colony PCR for AsCas12a expression vectors transformation.


We induce AsCas12a expression and purify it by gravity-flow column with Ni-NTA Sefinose(TM) Resin. SDS-PAGE and Western Blot is used to characterize protein purification as shown in Figure 2 and Figure 3.

Figure 2. The SDS-PAGE test of AsCas12a purification.
Figure 3. The Western Blot test of AsCas12a purification with Mouse-anti-His antibody.


We also determine the protein concentration with BCA kit.

Figure 4. The BSA standard curve.
BBa K2965021-Table 1. AsCas12a concentration..png


Finally, we test the activity of AsCas12a by detecting the fluorescence. A kind of 5bp single strand DNA with 5'-FAM and 3'-TAMRA is used as reporters in which the fluorescence of FAM can be quenched by TAMRA to display its DNA endonuclease activity. When AsCas12a is activated and cuts reporters, the cleavage of reporters will enable FAM to emit green fluorescence and neutralize the red fluorescence of TAMRA to be yellow. The extracted AsCas12a has ability to cut DNA compared to the control group and significantly higher activity compared to the bought AsCas12a.

Figure 5. The AsCas12a activity assay.

Improvement

iGEM Shanghaitech_China 2020 has improvevd AsCas12a (link:BBa_K3454046) in the way that two tags are added, namely MBP tag and His tag. MBP tag is maltose binding protein, which helps Cas12a expression, dissolution and folding in correct conformation. His tag can help purification Cas12a through Ni-NTA beads.

Figure 6. AsCpf1 western blot with histag.

We got our wide-type AsCas12a protein (link:BBa_K3454037) from BBa_K2965021. For higher expression yield in E. coli, we did codon optimization on the original coding sequence. Although the DNA sequences was changed, the amino acid sequence remained the same, so the protein was not changed after codon optimization. Then we introduced the selected mutations into WT plasmid through circular PCR reaction in the order of S542R, K548V, N552R and E174R, which is BBa_K3454038, BBa_K3454039 and BBa_K3454040, respectively. And for composite part with Histag and MBP tag: BBa_K3454041, BBa_K3454042 and BBa_K3454043. As a result, we successfully extracted three mutant proteins —— AsCas12a-R (BBa_K3454038), AsCas12a-RVR (BBa_K3454039) and AsCas12a-RVRR (BBa_K3454040). For more detail, see [1] page.

Reference

[1] Jeon Y, Choi YH, Jang Y, Yu J, Goo J, Lee G, et al. Direct observation of DNA target searching and cleavage by CRISPR-Cas12a. Nature Communications. 2018;9(1):2777-11.

[2] Mohanraju, P., Oost, J. v., Jinek, M. and Swarts, D. C. (2018). Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein. Bio-protocol 8(9): e2842. DOI: 10.21769/BioProtoc.2842.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 196
    Illegal PstI site found at 1258
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 196
    Illegal PstI site found at 1258
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 380
    Illegal BglII site found at 1922
    Illegal BglII site found at 2231
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 196
    Illegal PstI site found at 1258
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 196
    Illegal PstI site found at 1258
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
    Illegal NgoMIV site found at 3466
  • 1000
    COMPATIBLE WITH RFC[1000]


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