Evo-Engineered CRISPR-Cas9s Hit More Gene-Editing Targets
News Jun 24, 2015
One particular Cas9 nuclease, the one derived from the bacteria Streptococcus pyogenes, has excited the gene-editing world, but it has its limitations. While it is true that this Cas9, in a CRISPR-Cas9 system, can be teamed with different guide RNAs to seek different DNA sequences, it cannot always insert double-strand breaks near these DNA sequences. This Cas9, which is called SpCas9, must also recognize a specific protospacer adjacent motif (PAM), which may or may not sit beside the target DNA. Because DNA target sequences and PAM sequences are not always conveniently arrayed, SpCas9 sometimes fails to snip DNA with sufficient precision.
To get around this limitation, a team of Massachusetts General Hospital (MGH) scientists subjected S. pyogenes—and other bacterial species—to directed evolution, a positive-selection engineering system in which survival was enabled by Cas9-mediated cleavage of a selection plasmid encoding an inducible toxic gene. This engineering system allowed the scientists to rapidly evolve the ability of SpCas9 to recognize different PAM sequences.
From a collection of randomly mutated SpCas9 variants, the scientists identified combinations of mutations that enabled SpCas9 to recognize new PAM sequences. These evolved variants, the scientists estimated, essentially double the range of sites that can now be targeted for gene editing using SpCas9. Fortuitously, they also identified an SpCas9 variant that was less likely to induce the off-target gene mutations sometimes produced by CRISPR-Cas9 nucleases.
They also showed that the Cas9 nucleases that are derived from Staphylococcus aureus and Streptococcus thermophiles bacteria, not just S. pyogenes bacteria, can also function in the MGH team’s bacterial evolution system, suggesting that Cas9 nucleases from these additional bacterial can be functionally modified as well.
“We show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design,” wrote the authors. “These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis.”
“In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells,” the authors continued. “We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells.”
And so the MGH team, led by J. Keith Joung, M.D., Ph.D., assert that they can overcome the gene-editing limitations imposed by SpCas9’s preference for a certain PAM sequences—those in which a nucleotide is followed by two guanine DNA bases.
"[The additional evolved SpCas9 variant] with increased specificity should be immediately useful to all researchers who currently use wild-type SpCas9 and should reduce the frequencies of unwanted off-target mutations," Dr. Joung noted. "Perhaps more importantly, our findings provide the first demonstration that the activities of SpCas9 can be altered by directed protein evolution.”
“This work just scratches the surface of the range of PAMs that can be targeted by Cas9,” Dr. Joung added. “We believe that other useful properties of the enzyme may be modified by a similar approach, allowing potential customization of many important features."
Revolutionary Imaging Technique Uses CRISPR to Map DNA MutationsNews
The new high-speed AFM method can map DNA to a resolution of tens of base pairs while creating images up to a million base pairs in size. And it does it using a fraction of the amount of specimen required for DNA sequencing.READ MORE
AIDS Vaccine Design: New Data Offers Important InsightNews
New data published in Immunity further illuminate how some human beings generate powerful, HIV-blocking antibodies. The results offer important insight into a potential AIDS vaccine design.READ MORE
Comments | 0 ADD COMMENT
3rd Annual NGS Data Analysis and Informatics Conference
Feb 08 - Feb 09, 2018
3rd Annual Genome Editing & Engineering Conference
Feb 08 - Feb 09, 2018