Part:BBa_K4308023
CasΦ_crRNA(H4)
CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing[1]. For our target LICN00857, we designed some crRNA in our CasΦ system. And crRNA(H4) was used in the CRISPR-Cas system as the one with the lowest off-target rate among the RNAs we designed.
Biology
The insertion or deletion of uridine residues into kinetoplastid during RNA editing is a small non coding RNA that can pair with pre mRNA. crRNA edits RNA molecules, about 60-80 nucleotides in length, and is transcribed by a single gene. A hairpin secondary structure onto the spacer region of crRNA, which can increase the CRISPR-Cas system specificity for target cleavage[2], was added to H4 when designing.
Usage
By combining with CasΦ, H4 participates in the target recognition and cleavage of CasΦ system. Compared with normal crRNA, the specificity of CasΦ-H4 system increased.
Characterization
1. Effect of crRNA secondary structure on specificity of CRISPR-Cas system
As reported by Gersbach et al. a hairpin secondary structure onto the spacer region of crRNA can increase the CRISPR-Cas system specificity for target cleavage[2]. To verify whether this strategy is effective for the trans-cleavage activity of CasΦ-crRNA, we introduced different lengths of hairpin structures into the crRNA of CasΦ (Table 1). The FQ-reporter assays showed that the recognition of complementary target or mismatch target by CasΦ-crRNA was affected by the hairpin structure in crRNA at the same time. By comparing the specific signal from the complementary target and the non-specific signal from MT13, we found that crRNA with a 4-base pair stem structure (H4) may be able to improve the specificity of the CasΦ-crRNA system for target detection. (Figure 1)
Table. 1 The sequence of crRNAs with hairpin structures.
Figure. 1 The reaction rates of FQ reporter cleavage by Mut-4 with hairpin structure crRNA.
2. Performance of optimized system for DNA mutation detection
In order to verify the performance of our optimization CRISPR-Cas system for picking up DNA mutations from a large number of background sequences, we mixed different amounts of target sequences with mismatch sequences (MT13) to simulate artificial samples containing 50% to 0% DNA mutations. We chose Neg-K (a CasΦ mutant designed by our group) and crRNA H4 to construct the mutation detection system. As shown in Figure 2, compared with the conventional wild-type CasΦ - hairpin free crRNA system (WT/CrRNA in Figure 2), the Mut-4/H4 system showed better mutation detection performance. For Mut-4/H4, even if the fraction of target sequences was as low as 2%, the initial reaction rate of the fluorescence signal was still higher than that of the mismatch sequences.
Figure. 2 The reaction rates of FQ reporter cleavage with samples containing 50% to 0% DNA mutations.
References
[1] Pausch, P., B. Al-Shayeb, E. Bisom-Rapp, et al. CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science 369, 333-337, doi: 10.1126/science.abb1400(2020).
[2] Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, K. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat Biotechnol 37, 657-666(2019).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |