Difference between revisions of "Part:BBa K2965037"

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Fn-crRNA-HPV16-1

This part is a crRNA of FnCas12a, it is complementary to HPV16 L1 sequence. There is a 5' TTN PAM on the DNA strand opposite the target sequence. When FnCas12a is guided by this crRNA, once it recognize its target, it will be activated and exhibits a “collateral effects” of promiscuous DNase activity.

Usage and Biology

This part is a guide RNA for FnCas12a protein. It can combine with FnCas12a protein and help FnCas12a to recognize its dsDNA target. After the target be recognized, FnCas12a would exhibit a “collateral effects” of promiscuous DNase activity.

The CRISPR-Cas system is a prokaryotic immune system for acquired immunity. It can confer resistance to foreign genetic elements. CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences derived from DNA fragments of bacteriophages that have previously infected the prokaryote and are used to detect and destroy DNA from similar phages during subsequent infections, thus, the CRISPR arrays are comprised of a set of Cas genes and identical repeats interspaced by spacer sequences which is acquired from phages. CRISPR RNA (crRNA) is firstly transcribed as a precursor crRNA (pre-crRNA) from CRISPR arrays then undergoes one or two maturation steps to generate the mature crRNAs that function as guides in destruction of invading DNA or RNA. crRNAs can combine with Cas proteins to form an effector complex which recognizes the target sequence in the invasive nucleic acid by base pairing to the complementary strand and induces sequence-specific cleavage. FnCas12a is a member of class 2 CRISPR systems, thus do not form effector complex with other proteins, but work by itself. This part is a crRNA to guide FnCas12a and can help it to recognize a 22bp fragment from HPV16 L1 sequence.

Characterization

Results

DNA Cleavage Assays

In order to use Cas12a to detect HPV16 infection, we selected different target fragments from HPV16 L1 sequence (which is used for HPV typing), they are Target 1,2 and 3 (Figure 1). Fn-crRNA-HPV16-1 can specifically recognize Target 1 by base paring.

’’’ Figure 1. 500bp from HPV16 L1 gene.’’’Shows the primers we designed and targets (which is partly complementary with crRNA) for Cas12a to recognize. PAM is necessary for Cas12a to recognize the target, for FnCas12a PAM is TTTN.

Considering that our detection was carried out after RPA (recombinase polymerase amplification) and Cas12a with its crRNA would be added into the RPA mix. Firstly, we use FnCas12a and Fn-crRNA-HPV16-1 to explore the optimum ions concentration for Cas12a to make sure how much reaction buffer to add into the reaction mix.

’’’Figure 2. Doing DNA reporter cleavage assay with gradient ion concentration.’’’

We then compared 3 kinds of Cas12a each combined with 3 different crRNA (that can recognize Target 1, 2 or 3).

’’’Figure 3. The comparison between different Cas12a and different crRNA targeting different sequences.’’’Shows that crRNA targeting to HPV16 could not recognize HPV18 L1 sequence.

Fluorophore Quencher (FQ)-Labelled Reporter Assay

The results of DNA cleavage assays showed that FnCas12a combined with Fn-crRNA-HPV16-1 is more active than those with Fn-crRNA-HPV16-3 and Fn-crRNA-HPV16-2. In order to ensure this conclusion, we than quantitatively compared FnCas12a’s activity when combined with either Fn-crRNA-HPV16-2 or Fn-crRNA-HPV16-1 by using Fluorophore quencher (FQ)-labelled reporter. The result is showed in Figure 4.

’’’ Figure 4. Quantitatively compared FnCas12a’s activity when using Fn-crRNA-HPV16-1 or 2.’’’

The quantitative result illustrated that Fn-crRNA-HPV16-1 is just as capable as Fn-crRNA-HPV16-2 in guiding FnCas12a to HPV16 target.

Protocol

70bp DNA Reporter Cleavage Assay

Generally, 0.5μL crRNA (2μM) and 0.5μL Cas12a protein (1μM) were added directly into 7.6μL of RPA or PCR products with 0.4μL 10× Cas12a reaction buffer. Add 1μL reporter (70bp, 15μM), mix well to start cleavage. The reaction was incubated at 25℃ for 80min. Reactions were quenched with DNA loading buffer (30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol) containing 15 mM EDTA and separated by 1.5% agarose gel pre-stained with Gel red (TOYOBO).

Fluorophore Quencher (FQ)-Labelled Reporter Assay

The reaction mix was the same as DNA reporter cleavage assay. The only difference is that the reporter is a 5bp single strand DNA with 5’-FAM and 3’-TAMRA. For qualitative detection, an UV flashlight was used to check if FAM was emitted green florescence. For quantitative detection, a real-time fluorescence detection device was used, the reaction was carried out in a specially made plastic plate with a small groove. Florescence was measured every minute.

Sequence and Features