Increase targeting range to improve the usefulness of more compact CRISPR-Cas9

A method was developed by a team of investigators from Massachusetts General Hospital, short for MGH, to improve the usefulness and precision of the most common form of the gene-editing tools CRISPR-Cas9 RNA-guided nucleases. This method can be applied to Cas9 enzymes from other bacterial sources. The method was published in the online publication, Nature Biotechnology. The study evolves a variant of SaCas9 — the Cas9 enzyme from the Streptococcus aureus bacteria — that recognizes a broader range of nucleotide sequences. This allows targeting of genomic sites previously inaccessible to CRISPR-Cas9 technology. According to Benjamin Kleinstiver, PhD, a research fellow in the MGH Molecular Pathology Unit, the development of Cas9 variants with a broader targeting range is particularly important for applications requiring precise targeting of genomic sequences. What's more, the coding sequence of SaCas9 is twenty-three percent smaller than that of SpCas9. It is the version derived from Streptococcus pyogenes, which is a size difference that makes SaCas9 advantageous for potential therapeutic applications requiring delivery by viruses. Kleinstiver is also the lead and co-corresponding author of the Nature Biotechnology paper As we know that CRISPR-Cas9 nucleases are comprised of a short RNA molecule, twenty nucleotides of which match the target DNA sequence, and a Cas9 bacterial enzyme that cuts the DNA in the desired location. Cas9 needs to recognize an adjacent nucleotide sequence which is called a protospacer adjacent motif (PAM) during the match between the RNA and DNA sequences. In their studies published before in the Nature, the team from MGH showed a genetic system that enabled them to rapidly evolve SpCas9 to recognize different PAM sequences. The team was able to evolve versions of SpCas9 that recognize a broader range of PAM sequences, which doubles the range of targetable sites. The team turned to SaCas9 in their recent study. It naturally requires the PAM sequence NNGRRT - in which R can be either adenine or guanine and T must be thymine - adjacent to its target DNA. They were successful to develop a variant called KKH SaCas9 which recognizes PAM sequences with any nucleotide in the third position. The results increased the targeting range for two to four times. J. Keith Joung, MD, PhD is an associate chief of Research in the MGH Department of Pathology, professor of Pathology at Harvard Medical School, and co-corresponding author of the Nature Biotechnology paper. He is quite confident about this directed evolution approach. He thinks it can be used to modify the PAM specificity of SaCas9, which greatly expands the number of genomic sites that can be accessed by the important Cas9 nuclease. Targeting sites precisely is quite important for researchers who are involved in disrupting small genetic elements and DNA repair by homologous recombination. This method can provide an important blueprint for altering the recognition properties of the worth of Cas9 nucleases existing in many bacteria, according to Joung. Read more here:http://www.cusabio.com/Recombinant-Protein/Recombinant-Xenopus-laevis-African-clawed-frog-Transforming-growth-factor-beta-1-11090034.html