Part:BBa_K2965021

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

Result

We link AsCas12a fragment and pET-28a(+) plasmid to AbCas12a expression plasmid with His tag. Then we transfer 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 Biologocal Technology Institute sequencing.

Figure 1. AsCas12a colony PCR.

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 test protein purification as shown in Figure 2 and Figure 3.

Figure 2. AsCas12a SDS-PAGE.

Figure 3. AsCas12a WB.

We also determine the protein concentration with BCA kit.

Figure 4. BSA standard curve.

Finally, we test the activity of AsCas12a by detecting the fluorescent. The extracted AsCas12a has ability to cut DNA compared with the control group and is significantly better than the bought AsCas12a.

Figure 5. AsCas12a activity assay.

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]