Part:BBa_K2927005

LbCas12a

CRISPR Cas12a system is one of the bacterial adaptive immune systems. Cas12a protein is the RNA-guided enzyme that binds and cut DNA. When Cas12a protein bind with the specific crRNA, it will be activated. After Cas12a protein is activated, it will cut the target DNA as well as non-specific single-strand DNA (ssDNA), this certain function is used in our project. We designed six crRNA which is derived from the African swine fever virus (ASFV) to detect the DNA of the virus. This sequence is just a part of DNA sequence of the vp72 membrane protein of ASFV.

Reference
Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna, Chen et al., Science 360, 436–439 (2018)

Experiment Results

• Introduction

In our project, we combined three parts of biological reactions to detect ASFV specific sequence in samples. The first one is LbCas12a-crRNA system, which can specifically recognize ASFV specific double stranded DNA (dsDNA) sequence on P72 gene. The secondary part is the trans-activation of LbCas12a-crRNA system. When LbCas12a-crRNA system binds to ASFV specific dsDNA sequences, LbCas12a-crRNA system will cleave dsDNA and further degrade non-specific single stranded DNA (ssDNA). To detect the degradation of ssDNA in ASFV-activated LbCas12a-crRNA system, we will use the PicoGreen fluoresce dye to monitor the undegraded ssDNA, which is the third part. To transfer reaction from part I/II/III to detection, we plane to conjugate ssDNA on magnetic beads. The ssDNA conjugated magnetic beads will be easily captured and transfer by electromagnetic force. In the following result section, we will show our progress through experiments that supported our project design.

• Our targets

Steps to establish CRISPR-LbCas12a system

1. Expression of LbCas12a protein:

We transformed pHMT-LbCas12a into E.coli BL21, and then added 0.2 mM IPTG to induce protein expression (see notebook for details). The result showed that the LbCas12a protein expression in soluble fraction was induced by IPTG, and increased as time goes on (Figure 1). The predicted protein size of LbCas12a with MBP and His-tag is about 180 kDa, which is close to the induced protein indicted by red arrow in figure 1. We also examined the insoluble fraction of IPTG induced BL21 by SDS-PAGE, and confirmed that most LbCas12a protein was soluble (Figure 2).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1371
  Illegal BglII site found at 2108
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 3022
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1435