Part:BBa_K2644105

T7crRNADNMT（fnCas12a）

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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 22
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

Due to the diversity of mutation sites in human genes, not all mutation sites have suitable PAM (TTTN). However, Cas12a protein needs PAM to perform recognition function, so the detection of mutation sites is not completely detectable. Moreover, most mutation sites are very close to each other, and a crRNA can only detect one mutation. Considering different mutation sites in human genes correspond to different diseases, the detection is expensive. It is necessary to achieve one-time multi-locus mutation detection as efficiently as possible.

Biology

principle of designing crRNA：
First, we must put the loop structure which corresponds to the homologous crispr Cas12a as the 5’ side, then put the spacer which length 17-24nt to the 3’ side, the front end of the spacer must have PAM(TTTN) and the mutation side had best at the position of 2-7nt after PAM.

Reference

Li, Shi Yuan, et al. "CRISPR-Cas12a has both cis - and trans -cleavage activities on single-stranded DNA." Cell Research (2018).