saCas9, a "mini" CRISPR Cas9 nuclease that packs a lot of punch! Although the S. pyogenes Cas9 (spCas9) is the better known nuclease for CRISPR gene editing, the recent addition of a miniature Cas9 nuclease from S. aureus (saCas9) has many researchers excited about its potential to expand gene editing capabilities in vivo. Like spCas9, saCas9 is able to cleave double-stranded DNA. Unlike spCas9, saCas9's unique features give it an edge over its more popular cousin!
With the help of the CRISPR Cas9 system, scientists from Duke University can activate genes with the flip of a light switch! By attaching one portion of a light sensing protein to the CRISPR Cas9 system and the other portion to a gene activating protein, they were able to modulate genes by shining blue light on the cells. This has exciting potential applications in tissue engineering! #CRISPRCrashCourse #CRISPR
SaCas9 is just as efficient an editor as SpCas9 however it is only about three quarters the size of SpCas9. This makes it particular useful when it comes to in vivo delivery via adeno-associated viruses (AAV). In fact, scientists can fit not only SaCas9 but several gRNAs into the same AAV. #CRISPRCrashCourse #CRISPR #saCas9
Scientists from the University of California used the CRISPR Cas9 system to insert genes into mosquitoes to attack the malaria parasite, Plasmodium. This was such an effective technique that the mosquitoes carrying these genes had no parasites in their salivary glands at all! By inserting the entire CRISPR Cas9 system into the genome of the mosquito, they also created a gene drive, ensuring the parasite-killing genes are propagated to the next generations of offspring. #CRISPRCrashCourse…
Truncated gRNAs (tru-gRNAs) are just as effective as the full length gRNA in directing to its target, and even better at reducing off-target cleavage. This is because tru-gRNAs are more sensitive towards mismatches.
In order to achieve genomic editing at the nucleotide level, scientists attached a cytidine deaminase to a This hybrid protein enables targeted replacement of cytosines to uridines, which the cell can then read as a thymidine.
Harvard geneticist George Church is using the CRISPR system to insert mammoth genes for cold tolerance into the elephant genome. This will allow the elephants to survive better in colder climates, away from human activity.
Cas9 doesn't cut DNA just once, it cuts over and over again -- not stopping until its target site is disrupted. Because the cell introduces indels as it repairs these cut sites, this excessive cutting is not ideal for precise genetic edits. Scientists from Rockelleller University introduced mutations next to Cas9's target that will prevent Cas9 from cutting an already-edited gene, a method they call "CORRECT". #CRISPRCrashCourse #CRISPR
Cpf1 is the latest nuclease to join the CRISPR tool box. Not only is Cpf1 itself a smaller protein, it requires a shorter guide RNA, making it much easier to package into viral vectors such as AAV. Because it cuts so far away from its PAM site, it can undergo many rounds of cutting, allowing for a higher chance for genome editing via the HDR pathway. #CRISPRCrashCourse #CRISPR
CRISPR was used to knock out one of six PPO genes in a white button mushroom so that it can resist browning. This mushroom is the first CRISPR-edited organism to leap over regulatory processes and perhaps into grocery stores in the near future.
Scientists from University of Massachusetts light up the genome with CRISPR gRNA-GFPs. They engineered RNA to GFPs of the primary colors (red, green, or blue) and achieved additional labels of cyan, magenta, and yellow with combinations of GFPs. By combining all three GFPs, they achieved a seventh label, white. #CRISPRCrashCourse #CRISPR