The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations

Gantz V.M. and Ethan Bier. Science (2015) 348:442-444.

Zoonotic diseases, such as malaria and West Nile, result in illness and can be fatal.  Traditionally these diseases have been controlled by individuals wearing insect netting and by reduction in insect populations.  With the advent of gene editing techniques such as CRISPR/Cas9 systems so called “Gene Drives” have become a third possibility.  Gene Drives involve the selective gene editing and propagation throughout a population to prevent carrier infection.  In order for a Gene Drive to be successful both copies of a gene must be edited in the modified individual to ensure transmission to all offspring.  Gantz and Bier have now demonstrated the ability to use the CRISPR/Cas9 system to edit both alleles in somatic and germline cells through a process called mutagenic chain reaction (MCR).  In Drosophila this involves insertion of the CRISPR/Cas9 machinery through Homology Directed Repair into the target genome which allows for continuous editing of the target site through insertion of the CRISPR/Cas9 machinery.

Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients

Schwank, G. Cell Stem Cell (2013) 13:653-658.

CRISPR/Cas9 systems hold promise as a therapeutic gene therapy for a variety of chronic genetic diseases.  Cystic Fibrosis is the result of mutations in both alleles of the CTFR gene and is currently not a treatable condition.  In this study the authors present results demonstrating the ability of a CRISPR/Cas9 system to replace the mutant copy with a functional allele through homologous-end-joining repair in cultured small intestine cells of a cystic fibrosis patient.  The specificity of CRISPR/Cas9 systems could allow for gene therapies to cure genetic diseases.

Conformational control of DNA target cleavage by CRISPR–Cas9

Sternber, S.H. et al. Nature (2015) 527:110-113.

One of the major obstacles facing CRISPR/Cas9 systems is off-target cleavage events.  Understanding the mechanisms by which CRISPR/Cas9 systems identify and cleave DNA could provide insights into ways to decrease off-target cleavage.  While Cas9 crystal structures have been elucidated the HNH domain active site was found to be ~30Å away from the targeted DNA thus preventing a detailed understanding of the cleavage mechanism.  By developing a Förster resonance energy transfer (FRET) based system Sternber et al were able to determine that two α-helices induce a conformational shift that acts as a signal transducer to activate both the HNH and RuvC nuclease domains thus coordinating cleavage of both DNA strands.  Furthermore, the authors determined that Cas9 could cleave off-target sites that contained only 1-3 mismatches a the distal end of the guide RNA since the activating conformation shift was still able to occur.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Kleinstiver, B.P. et al, Nature (2015) 523:481-485.

While the construction of unique guide RNAs theoretically allows CRISPR/Cas9 systems to target any unique genomic sequence the range of possible sequences is limited by the need for a specific protospacer adjacent motif (PAM).  For instance, the widely adopted Cas9 endonuclease requires an NGG PAM to directly follow the guide RNA sequence.  To overcome this limitation Kleinstiver et al engineered Cas9 variants to recognize novel PAM sequences using a directed evolution approach thus expanding the range of sequences CRISPR/Cas9 systems can be used to edit.

Scientists Seek Moratorium on Edits to Human Genome That Could Be Inherited

Nicholas Wade, The New York Times, December 3rd, 2015.

An international panel called for a pause in using CRISPR/Cas9 technology to alter genes that could be inherited by future generations.  This moratorium was the result of a recent international meeting held at the National Academy of Sciences in Washington, jointly called by the National Academy of Sciences, the Chinese Academy of Sciences, and the Royal Society of London in response to the use of CRISPR/Cas9 technology to alter a non-viable human embryo earlier in 2015.  While the moratorium does not have regulatory authority, it is expected most scientists will follow the new guidelines.  The respective scientific bodies did leave the door open for removal of the moratorium, stating the issue “should be revisited on a regular basis.”

MIT, Broad scientists overcome key CRISPR-Cas9 genome editing hurdle

News Office, MIT, December 1, 2015.

One of the major obstacles facing wider adoption of CRISPR/Cas9 systems as a gene editing and gene therapy technique is the off target cleavage of DNA.  In a report published in Science ( MIT researchers used rational protein engineering to improve CRISPR/Cas9 specificity.  Using the crystal structure of the CRISPR/Cas9 complex researchers identified a positively charged groove that is able to stabilize binding of negatively charged DNA.  By changing three of the positive amino acids found in this groove to neutral amino acids the researchers reasoned that DNA binding would become more dependent on proper Watson-Crick base pairing between the CRISPR guide RNA and the targeted DNA.  This improved Cas9, coined “enhanced specificity” SpCas9 or eSpCas9, eliminated 22 of the 24 off-target events observed when compared to wild-type Cas9 treatment.

New Edits to Animal Genes Cut Down on Rough Drafts, but Not on Worries

Amy Harmon, New York Times, November 26th, 2015.

Gene editing technology, such as CRISPR/Cas9 and TALEN systems provide the opportunity to edit not only the plants humans consume but animals as well.  Gene editing technology is currently being applied to the cattle industry to create hornless dairy cows in order to prevent farmers from needing to dehorn their livestock.  More immediately consumers will have access to salmon that has been modified in order to substantially increase its growth rate.  Gene editing is also being used for therapeutic goals with pigs being altered to grow human organs that will not be rejected and to make malaria resistance mosquitoes all in an effort to improve human health. While these technologies provide many opportunities some scientist and activists are promoting caution while the FDA has yet to release guidelines for gene editing.  In order for gene editing technology to become widely adopted scientist, the public, and government regulators will need to come to a consensus as to the extent to which this technology is used.

CRISPR/Cas patent wars have begun at the European Patent Office

Dr. James Legg,, November 23, 2015.

The battle for patents related to CRISPR/Cas9 technology is beginning to heat up.  On February 11, 2015 the first CRISPR/Cas9 European patent was granted to the Broad Institute at MIT and Harvard and by October 26 nine different groups have filed oppositions.  The fight over the CRISPR/Cas9 patents extends to the US where ~13 patents have been granted.  In both the US and Europe these disputes are based around who invented the technology first with competing claims coming from both the University of California and the Broad Institute.  With CRISPR/Cas9 technology opening up possibilities in agriculture and gene therapy this fight is set to continue for years to come.

Novel technology vastly improves CRISPR/Cas9 accuracy, November 18, 2015.

In an effort to improve CRISPR/Cas9 targeting research at the University of Massachusetts Medical School combined the old with the new. Zinc Finger DNA editing was originally developed in the early 1990’s by combining the DNA binding Zinc Finger protein domain with the Fok1 nuclease domain.  Since then researchers have developed tools to design zinc-finger binding domains to target specific DNA sequences.  The researchers at the University of Massachusetts combined this ability with the new CRISPR/Cas9 system by fusing a Zinc Finger domain to Cas9 to enhance the specificity of DNA targeting.  By combining these two different DNA targeting technologies the risks of off target effects.

‘Designer cells’ reverse one-year-old’s cancer

James Gallagher, BBC, November 5, 2015.

Genetic therapy has reached a new milestone with one-year-old Layla seemingly cured of childhood leukemia.  Layla was recently diagnosed with incurable leukemia and as a last ditch effort her parents and doctors received permission to try an experimental gene therapy treatment.  Donor immune cells were edited with TALENs to seek out and kill only the leukemia cells in Layla’s body.  Only a few months after the treatment Layla had no traces of leukemia in her body.  While this treatment used an older, more expensive gene editing technique known as TALENs the advent of the cheaper and easier CRISPR/Cas9 technology may make more treatments like this a reality.