New Applications in CRISPR – What to Look For!

Cold Spring Harbor Laboratory is a research utopia built amongst the trees and wildlife in peaceful settings about an hour from New York City. Home to eight Nobel Prize winners, the institution has sat at the forefront of cancer research, molecular genetics, biomedical sciences, and DNA chemistry for the past 100 years. A perfect setting then for a meeting of the greatest minds working on the hottest genome engineering technology of the decade.
Genome engineering: The CRISPR-Cas Revolution” was a focused deep dive into what’s new in genome engineering, held over four days in late July. In her opening address, Jennifer Doudna, Professor of Chemistry and of Molecular and Cell Biology at the University of California, Berkeley, discussed how CRISPR is a world-changing technology born from many years of scientific investigation into bacterial immune systems.
It was her lab that first showed that a tool from the bacterial immune system called Cas9 could be used to precisely target and edit a gene of interest when combined with a synthetic guide RNA. Overnight, CRISPR became the most powerful tool for editing, manipulating, and investigating the blueprints for life.
The conference showcased the cutting-edge of CRISPR research, with talks from the thought leaders and the architects of the field. Twist Bioscience attended Cold Spring Harbor as event sponsors sharing in the excitement. Below are our top picks for up and coming CRISPR technology showcased at the conference that are set to take the field by storm over the next year.
Single base editing with CRISPR
Activation-induced cytidine deaminase (AID) is an enzyme vital for antibody maturation facilitating the hypermutation of an antibody-encoding DNA sequence by converting cytidines (C) to thymidines (T). This base flipping activity allows our immune system to learn how to defend against new pathogens.
In cells, AID is precisely controlled to only work on antibody encoding sequences as to not cause mutations throughout the genome. However, if we could control the activity of AID single base pair genome modifications could be made in any DNA sequence of interest. DNA sequences could be mutated one base at a time allowing the precise study and modification of gene function, and diseases caused by single point mutations could be simply modelled in laboratory cell lines.

Artist’s rendition of blood cells in a patient with sickle cell anaemia – a disease of the blood caused by a single point mutation.

For the first time, CRISPR makes this tool possible. A mutant of Cas9 called “dead Cas9” has its blades blunted, causing the enzyme to target specific sequences but leave the DNA uncut. The activity of any DNA acting enzyme can now be precisely controlled by fusing it to dCas9. 
Fusing dCas9 to AID has been shown to mutate single cytidines into thymidines in a targeted fashion. At the conference, presented applications for this technology included drug discovery, gene disruption, and the engineering of mouse models for diseases caused by point mutations.
The holy grail for single base editing will be to develop a suite of single base editing tools. Currently with AID, only C->T is possible. Through either protein discovery or protein engineering, expect editors for other bases being developed in the near future.
Precisely controlling CRISPR from the outside
A typical CRISPR experiment involves transfection of the Cas9 protein, the guide RNA, and any other payloads of interest directly into the cell.
This is already a powerful tool, however, there is a missing dimension to the experiment – time. Currently it is onerous to switch CRISPR on and off in a cell environment, however the ability to do so would make gene editing highly tuneable, affording tight precision in genetic studies with editing that is active only exactly when needed. 
By extension, if CRISPR was to be used therapeutically, temporal control would allow genetic engineering to be administered with pinpoint accuracy before being turned off again so there is no risk of lasting effects from residual Cas9 in the cells.
Anti-CRISPR molecules that inhibit Cas9 are the first step towards realizing a CRISPR on/off switch. CRISPR originates from the bacterial immune system, protecting the bacteria from bacteriophage infection by cleaving viral-origin nucleotides. Just as bacteria can become resistant to antibiotics, evolution dictates it plausible for bacteriophage to build a resistance to CRISPR in order to survive and propagate.

Anti-CRISPR proteins are part of a developing toolbox that will allow us to switch CRISPR on and off exactly when required, a bit like a light switch.

AcrIIA4 is one such anti-CRISPR protein invented by viruses. AcrIIA4 fits perfectly into the DNA binding pocket of Cas9, clogging the enzyme up, allowing the viral-origin DNA to survive. Application of this enzyme is an efficient and elegant off switch for Cas9.
By virtue of the arms-race for survival between the bacteria and the bacteriophage, it is imaginable that bacteria challenged by viruses with anti-CRISPR proteins have evolved a set of anti-anti-CRISPRs to survive. At the time of writing, there are no anti-anti-CRISPR proteins reported. Yet their discovery, if they exist, will provide the other half of the on/off switch - affording complete temporal control over genome editing with CRISPR.
Multiplex genome control with Cpf1
Cas9 is not the only CRISPR associated enzyme able to precisely cut DNA by using RNA as a guide. Cpf1 is another key enzyme that has seen considerable use over the last year in the study of complex disease. In the study of complex diseases that are caused by multiple simultaneous genetic perturbations like cancer, a single site of genomic engineering is not enough to build an effective model. To study such problems, multiple genes need to be edited at once – a process called multiplexing.

An illustration of a cancerous legion on the skin. Multiplexed genome editing allows researchers to model diseases like cancer that are caused by multiple simultaneous genetic perturbations.

In a natural CRISPR system, a swathe of enzymatic machinery processes the gRNA and feeds it to Cas9. Researchers have simplified this system with synthetic guides that look identical to processed guides. Cas9 can handle one 100-base guide at a time, to cut one sequence at a time.
Expressing multiple guides to target multiple regions is cumbersome, requiring intervention with nucleases or multiple promoters to process the guides for Cas9. On the other hand, Cpf1 guides are much shorter at 40 bases, and the enzyme contains its own processing machinery.
This means Cpf1 multiplexing plasmids are considerably trim in comparison to equivalent Cas9 plasmids, allowing for easy packaging into lentiviruses for delivery into mammalian cells. Cpf1 is picking up speed in the community, especially with the release of a “dead Cpf1” and functional fusions between Cpf1 and DNA acting proteins. With Cpf1’s ability to edit multiple genetic regions simultaneously, expect to see it become a vital enabling tool advancing the research of complex diseases in the near future.
“Genome Engineering: The CRISPR-Cas Revolution” was an excellent conference, showcasing the exciting state of ever expanding CRISPR research. As our understanding of CRISPR grows, world changing applications will roll in regularly, and we will see the technology applied to some of society's biggest challenges - including the treatment and understanding of complex and devastating diseases.Opening image: The laboratory at Winter. Source: Wikicommons. Author: AdmOxalate