No, we are happy to design the guide RNAs and perform the assay on small or large fragments.
The smaller your fragment the less target sites we have to work with, however if you have a small specific site you want to target this may be the best option for you. We standardly test from 5 gRNAs, however if you only have a few guide RNA targets within your fragment, please contact us and we can discuss how best to proceed.
We have so far run the assay with insert sizes up to 5kb. If you have a very large fragment to test feel free to contact us and we can discuss how best to design the assay for your target sequence.
We currently run the assay with Cas9 and Cas12a, depending on factors such as GC content of your target sequence and experimental conditions such as temperature, we can help you choose which nuclease to use.
This depends on the availability of the recombinant nuclease and ownership of its intellectual property. If you would like us to run our assay with a nuclease other than Cas9 or Cas12a, please contact us and we can look into it for you.
This will partially depend on the complexity of your project and factors such as plasmid preparation and number of guide RNAs tested. However, as an example, you can expect the design, production and testing of around 10 guide RNAs for a non-complex target sequence to take around three weeks once we have the target plasmid.
The assay results in your target plasmid being cut to varying degrees depending on the cutting efficiency of the tested guide RNA. We visualise this on a gel (see our assay) and use band intensities to work out the percentage efficiency.
We will provide you with the gel image as well as the calculated efficiency:
If the guide RNA does not cut we only see one band relating to the plasmid.
If the guide RNA cuts efficiently we see two bands relating to the two halves of the cleaved plasmid.
If the guide RNA cuts but with a lower efficiency we see three bands; one relating to the uncut plasmid and the other two to the cleaved plasmid.
As genome editing also depends on the methods at your end (e.g. transformation method, selection method, correct storage of the guide RNA, screening and capability of your species to induce mutations through its DNA repair mechanisms) we cannot guarantee that our guide RNAs will result in a positive genome editing event. Furthermore, if you have chosen a region in the genome with physical barriers such as high levels of heterochromatin this can also reduce the efficiency of the CRISPR-Cas complex. What we have seen however, is that some guide RNAs do not cut at all under certain conditions or even under optimal conditions. Our service aims to identify the best guide RNAs in-vitro for you to try in-vivo and remove the chance of you attempting to edit targets which will not work under your experimental conditions, even in a test-tube.
We have only seen this happen when testing very small numbers of guide RNAs which is why we only test from 5 guide RNAs. We find that the more guide RNAs we test, the better chance we have of identifying ones with a good cutting efficiency.
Potential reasons for not identifying efficient guide RNAs include:
The insert in the plasmid does not match the sequence of the target gene/ region sent to us for guide RNA design. For this reason, we ask you to sequence your target plasmid before sending it to us or if you have asked us to clone your insert into a vector for you, we will sequence the plasmid before performing any assays.
Your experimental conditions are likely to result in very low cutting efficiencies. If you request our services and we think there may be issues, we will discuss this with you when building a quote to make sure our assay is a good fit and to help you choose an appropriate number of guide RNAs.
If we cannot identify a clear reason why none of your guide RNAs cut, we will re-run the assay, along with positive controls to ensure that our reagents are functional. If you want us to make and test additional guide RNAs, as a not-for-profit service we will need to charge an additional fee.