Researchers have overcome a major limitation of the CRISPR-Cas9 gene editing system, which should allow previously inaccessible parts of the genome to be edited with ease.
What is CRISPR-Cas9?
Clustered regularly interspaced short palindromic repeats (CRISPR) is used in CRISPR-Cas9 and CRISPR-associated protein, a gene editing system that has created considerable excitement in the last few years. This is because it is faster to work with, cheaper, more accurate, and typically more efficient than older gene editing approaches.
Believe it or not, CRISPR-Cas9 was originally discovered as a naturally occurring gene editing system found in bacteria, which was borrowed for use in science. In their natural environment, the bacteria capture pieces of DNA from invading viruses and then use them to make DNA segments called CRISPR arrays. These arrays allow the bacteria to memorize the virus so that if the same or similar ones attack again, the bacteria can defend themselves by using the arrays to produce RNA segments that target the DNA of the virus. The bacteria then use Cas9 or a similar enzyme to cut the target DNA apart, thus killing the virus.
Researchers use this natural gene editing system in a similar way in the lab by creating a small piece of RNA that has a short “guide” sequence that binds to a specific target sequence of DNA within a genome. This RNA also binds to the Cas9 enzyme. Using the same method as the bacteria, researchers use this modified RNA guide to identify the DNA sequence, and then the Cas9 enzyme cuts the DNA at the desired location on the genome. Once the cut is made, the researchers then use the cell’s DNA repair machinery to add or delete pieces of genetic material or even make changes to the DNA by replacing a segment with a modified DNA sequence.
Making the best even better
One of the limitations of CRISPR-Cas9 is the requirement that there be a particular short DNA sequence present at the target site on the genome. For CRISPR-Cas9 to work, the Cas9 protein seeks out a short region in the viral DNA called a protospacer adjacent motif (PAM). PAM sequences are fairly common in our DNA, which means that scientists have been able to make use of CRISPR-Cas9 to edit most genes; however, some cannot be targeted as they are not near one of these PAM locations, and this has been a real hurdle.
However this may no longer be an issue thanks to a team of researchers at Massachusetts General Hospital who have modified the system to be less dependent on this requirement, allowing them to target any location on the entire genome. In a new study published in the journal Science, the researchers were able to overcome this limitation .
The research team accomplished this by creating variants of the Cas9 protein that do not require a specific PAM in order to bind to and cut DNA. They created two variants, SpG and SpRY, which allow DNA editing at an efficiency level previously not possible using the regular Cas9 enzyme.
The new approach allows targeting of previously inaccessible regions of the genome by greatly reducing the necessity for the enzymes to identify a PAM. This opens the door to some exciting possibilities, as the entire genome is now targetable and open to interventions in the context of combating diseases.
The next step for this line of research is to develop a deeper understanding of the mechanisms involved in these Cas9 protein variants and exploring their potential.
Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG capable of targeting an expanded set of NGN PAMs, and further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN>NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.
This research has overcome a major limitation to the CRISPR-Cas9 gene editing system and paves the way for some real innovations in the world of gene editing and modification.
 Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020). Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science.