Opening wider genomic access with a flexible CRISPR enzyme

Pranam Chatterjee and Noah Jakimo, Molecular Machines group

The CRISPR-Cas9 system has proven to be a versatile tool for genome editing, with numerous implications in medicine, agriculture, bioenergy, food security, and beyond. The range of targetable DNA sequences is limited, however, by the need for a short sequence of DNA beside the target site, called the PAM. In total, there are only a handful of CRISPR enzymes with a short enough PAM sequence to be able to target a large portion of the total DNA in a genome. In this study, we identify a natural Cas9 enzyme from the bacterial genome of Streptococcus canis that has a PAM sequence with only a single G as its PAM sequence (5’-NNG-3’), allowing flexible targeting of up to 50% of all DNA sequences in living organisms. This new molecular tool potentially grants unprecedented access to correct disease-related mutations, enhance agricultural methods, and expand research efforts. 

FAQ: Opening Wider Genomic Access with a Flexible CRISPR Enzyme

  1. What is the problem to which this study poses a solution?

    Right now, gene editors are not able to access a large portion of all DNA sequences with ease, constraining various efforts. Our enzyme, which only needs a single DNA base to be next to the target sequence, allows access to up to 50% of DNA sequences. 

  2. What exactly does the newly identified enzyme do, and how?

    The enzyme is a Cas9 molecule, which can be guided to a specific DNA sequence to generate a modification, such as a mutation (to silence a gene), an insertion of DNA, or conversion of a single base. With a short required PAM sequence, it can perform these activities at nearly 50% of DNA sequences.

  3. How did you identify this enzyme?

    Basically, CRISPR is a method that bacteria use to remember which viruses invaded it, by storing parts of the virus in its own DNA, and then activating the Cas9 enzyme to chop up the viral DNA when it gets infected again—it’s essentially an immune system.

    As computer scientists, researchers Pranam Chatterjee and Noah Jakimo harnessed this knowledge to create a computational pipeline by identifying the short sequence beside viral DNA sequences that the bacteria’s CRISPR system wants to chop up to protect itself. Those short sequences are the predicted PAMs for the Cas9 enzymes from those bacteria. The team calls this pipeline the Search for PAMs by ALignment Of Targets (SPAMALOT). The results from this data-mining exercise predicted that the Cas9 from Streptococcus canis may potentially have a new and short PAM sequence. 

  4. Why hasn’t this been done before?

    Most labs are much larger than the Molecular Machines group and are funded to do large-scale experiments. They end up using exhaustive experimental methods to engineer new proteins, which takes a lot of time and effort. As computer scientists, we wanted to make this a big data problem that can be solved primarily computationally and simply require experimental validation. 

  5. There’s a lot of concern about how CRISPR might inadvertently damage the natural ecosystem, cause irreparable evolutionary harm, or be used for nefarious purposes. Can you speak to how your improved CRISPR technology influences, and is influenced by, these concerns?

    We are confident that the advent of new CRISPR technologies, like our new enzyme, are key additions into the CRISPR toolbox, which needs to be maintained in a safe and effective manner. We hope that these additions will further enable other scientists to validate which ones are the most useful for advanced purposes. At the same time, we have introduced a potentially dangerous new tool. It’s up to us to communicate both the benefits and the possible harms of this technology, especially with its increased targeting range. 

  6. Who is this technology for? Who do you want to use your more efficient CRISPR technology, and how?

    Primarily, this enzyme will be most useful to modify very specific locations of DNA, such as disease mutations. It’s easy to go to anywhere in a gene and silence it. But for specific base editing purposes, our new enzyme will potentially be very useful due to its flexibility of targeting. 

  7. What are the greatest challenges to implementing this technology in the real world?

    We have still yet to see the results for the first clinical trials using the standard CRISPR-Cas9 system. However, agriculture and various other industries have already implemented CRISPR-Cas9 and have seen high levels of success. The greatest challenges, however, are to minimize off-target effects (going to the wrong DNA sequence) and ensure high efficiency of editing. 

  8. Has this technology been proven effective? Are there any studies or pilot programs that demonstrate its use?

    We are beginning collaborations with various groups, such as the Long Lab at NYU as well as identifying licensees in pharmaceutical and biotech companies to accelerate the development and use of ScCas9 in preclinical experiments. Specifically, we are interested correcting mutations of genetic diseases such as Duchenne’s Muscular Dystrophy, which remains partly intractable due to the lack of flexible targeting with the standard Cas9 enzymes.