Please review the proposed puzzles for the initial OpenCRISPR round

Nando has create a set of 28 puzzles that meet Johan’s approval in terms of the experimental process.  But before formally launching the challenge, we would like to make sure they are all reasonable from the perspective of players solving them in silico.  And for that we need everyone’s help.

If you’re willing and able to help, I suggest starting by picking one of the 4 projects at random (so everyone isn’t working on the same puzzles) and try solving multiple (or all) the puzzles in that project (but not necessarily from top to bottom).  As you solve puzzles, post the results here.  I guess I’ll create a spreadsheet to keep tally of which ones have “approval” or have flags raised. I’ll post again when I’ve done that.

This should be a very interesting challenge, from many points of view!


Hi Omei, just wondering what you wanted included as results. Thanks



No need to provide a specific solution.  We’re mostly looking for a Yes, it seems like a reasonable puzzle, or something about it that makes you question it.  For example, you found it unusually hard to find any solution, or it seemed like there would be a limited number of (essentially different) ways to solve it.

If you want to go beyond that and suggest a way to improve it, that’s welcome, too.

On second thought, maybe it would be good to include the solution sequence, so I can at least spot-check that we have a common understanding of which puzzles we’re talking about.

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@whbob, the current set of proposed puzzles doesn’t use Trp as an aptamer.  If you follow the link in the original post, do you get to a Trp puzzle?  If so, can you give me the URL of the puzzle so I can figure out where the confusion is?  


TEP 1 - A series

CRISPR/Cas9 - TEP 1a - ON

Ugly solve. Has MS2 positioned more like in an OFF switch


CRISPR/Cas9 - TEP 1a - OFF

Quick and ugly solve. Can solve in an OFF switch style with MS2 next to the aptamer as I prefer. However this solve is pretty sensitive to whatever else I do to break the way too many a’s in line.


TEP 1 - B series

I think the B series has better shot at doing well than the A series. Was far easier solving. I think the aptamer is optimal oriented for a better switching options.

CRISPR/Cas9 - TEP 1b - ON

Can be solved following blueprint. Having MS2 straight opposite the THEO aptamer, which is typical for an ON switch. Follows blueprint. Can be made symmetric.


CRISPR/Cas9 - TEP 1b - OFF

Can be solved following blueprint. Having besides the the THEO aptamer, which is typical for an OFF switch.


I think the Theopylline aptamer wants traditional orientation. This is the way it turns when it is positioned in the sequence as a continous sequence. (Unsplit) It has the bigger internal loop pointing towards the switching area. I think it wishes to turn that way too, when it is split also.

Can the A series with the inversed aptamer be make working, probably. But I think the one particular orientation of the aptamer will be easier and better.

So basically I think the TEP puzzles looks fine. Except for the A’s series. I would still love to see how they do.

Additional TEPs

CRISPR/Cas9 - TEP 2 - ON

Ugly solve


Just for the fun of it, I also tried place in MS2 as a split hairpin and treat it just as if it was an aptamer. It will probably not be too fond of it in practice, since the small hairpin loop is exacly where the MS2 virus protein binds. And that is the loop I’m splitting. 

My intention however was to be allowed to solve this puzzle in my favored blueprint style. So here I let the MS2 take the aptamer spot - because the natural MS2 spot are already taken by the Theo aptamer.

In the best FMN/MS2 labs (Same state NG 2 and Exclusion NG2), the whole puzzle were akin to a balloon. A neck was holding a sequence bubble - wherein the switch elements like the MS2 and FMN aptamer tended to prefer being placed in rather specific ways. The FMN aptamer was attached to the neck and the MS2 was placed right across the FMN aptamer in the Same state case, or right beside it in the Exclusion case.



Exclusion style blueprint with MS2 next to the aptamer


That was a typo, sorry.  I selected the CRISPR/Cas9 - TEP 1b - ON lab challenge.
In each of these, I created a stem above the locked bases using U’s until the stem locked.  I introduced the MS2 at about base 70 and slid it down until state 1 and state 2 met the constraints.  Then I populated the loop with G’s to meet the A limits.

RE: TEP-1b-ON: The bases were so overlaid on my monitor that I couldn’t tell where the target bases should be.  I could not meet the target shape.

Controls are essential for quality experimentation so I started there. I quickly obtained a sequence for CRISPR/Cas9-Control ON


   I pasted this same sequence into the CRISPR/Cas9-Control ON (freestyle) puzzle as well [].


    I began the CRISPR/Cas9-Control OFF puzzles as well  [ and] and noticed several things about these four puzzles:

1)    The Oligo (Target DNA: CUGCGUAUUUCUACUCUGAG) is bound in every single state which confers -39.3 kcal of energy (through bonds), yet there appears to be a penalty of 28.22 kcal listed where the energy from the Oligo would normally appear.

2)    Having pasted the exact same sequence into each of the four puzzles I looked at the Total Energy listed (sans the 28.22 kcal) and found that for the CRISPR/Cas9-Control OFF, OFF (freestyle), and ON (freestyle) the number provided (in white) was -114.2, while for the CRISPR/Cas9-Control ON the energy given was -113.8. The difference comes from a single bond (GU) which is formed ( ONLY in Control ON ) between bases 21 and 131. This bond adds -1.4 kcal of energy to the stack while causing the energy in the open “hook” to be -1.5 kcal. This bond does not form in the other three puzzles where the unpaired bases make the hook energy -5.4 kcal.

3)    In the Control OFF puzzle one of the criteria is that the, “MS2 hairpin must not form in state 1” (despite the puzzle not actually being a switch with Control ON [is that the plan?]), yet the outlined structure that the RNA is required to fold into INCLUDES THE MS2 HAIRPIN! Thus, if the MS2 sequence (ACAUGAGGAUCACCCAUGU) is pasted in the same place as in Control ON puzzle, then this puzzle is unsolvable. Luckily the puzzle is possible if we stamp the MS2 hairpin elsewhere in the puzzle. The following sequence demonstrates how CRISPR/Cas9-Control OFF may be solved: GACGCAUAAAGAUGAGACGCGUUUGAGAGCUAAUAAUAUGUGAUAUAUGAAAAUAUAUUGUCCUCAUGUUAGGGAAACCAACAUGAGGAUCACCCAUGUGAAUGGGUGGCAUAUUAUUAGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU.

4)    However, the stamper tool pastes more than just the MS2 sequence.

        Here are the sequences stamped if one were to click on the same base with the stamper tool repeatedly: 1st stamp=ACAUGAGGAUCACCCAUGU, 2nd = ACAUGAGCAUCAGCCAUGU, 3rd = ACACGAGGAUCACCCGUGU, 4th= ACAAGAGGAUCACCCUUGU, 5th= ACAGGAGGAUCACCCCUGU, 6th= ACCUGAGGAUCACCCAGGU, 7th= ACCUGAGGAACACCCAGGU, 8th= ACCUGAGGAUCACCCGGGU, 9th= ACCUGAGGAUCACCCUGGU, 10th= ACGUGAGGAUCACCCACGU, 11th= ACUUGAGGAUCACCCAAGU, and so on. Clicking incessantly the MS2 sequence will be stamped every 25th click or so [will say, “present in (1)” in the objective box].    

   That’s all I had time to do thus far, but hope to try out the rest of the puzzles later on. 

TEP 1b OFF (not very inspiring solve)


Can you elaborate on your overlay problem with TEP-1b-ON?  I’ve had this problem with other puzzles, but not this specific one.  I’m wondering whether different players are seeing different things.   Here’s what it looks like for me:

Thanks for the detailed comments, @cynwulf28.  I’ll try to address them.

Re 1 and 2, there are known quirks/defects in the  energy model.  There are also three different energy models, that produce three different results.  So I guess my question is whether there is anything in 1 and 2 that you think is a significant impediment to solving these puzzles?

Re 3, the purpose of the puzzle is to create solutions that contain the MS2 sequence, but the sequence is bound up in some other structure (which is what should happen for an OFF puzzle.)  So I think everything you said is accurate except for “Luckily”. :slight_smile:

Re 4, congratulations! You’ve discovered a little-advertised feature of the stamper for stamping in other variations on the MS2 sequence.  Research from the Greenleaf lab indicated that these variations bind to the MS2 protein almost as well or even a little better, than the default MS2 sequence.  I know some players (including me) have experimented with using them, but I’m not sure if anyone has published any conclusions about them.

I was using the non-target mode. That’s what confused me.

Seems to be a lot of potential variability for solutions.

FMN 1a - OFF
Again, appears to be a lot of potential variability for solutions.

Here’s a compact skeletal solution for Tetracycline inv - OFF


… and a similar one for Tetracycline inv - ON