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.

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.

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]