Safety is considered one of the most important aspects in synthetic biology and a big concern is biosafety, this year our team expanded this aspect through Predicting novel brake elements enabling more controllable & safer CRISPR Technology for gene editing and gene expression regulation as you know gene editing technology has many applications in medicine including gene therapy, immunotherapy and limiting drug resistance. All these fields mainly depend on a powerful method using the “CRISPR-Cas system”.which sparks our imagination about what could happen if these gene editing tools get out of control and end up in gene mutations rather than gene repairs. We herein, introduce a new panel of anti-crispr proteins (acr) that can be used to counteract the cas12g action and cleavage function.

Anti-CRISPR: Anti-(Clustered Regularly Interspaced Short Palindromic Repeats) or Acr is a group of proteins found in phages that help viruses to inhibit the normal activity of CRISPR-Cas, to evade the immune system of certain bacteria which are natural OFF-switches for CRISPR-Cas

There are different families of Acr that work on different levels and are highly diverse but most of them block CRISPR activity by one of three ways:
Inhibit crRNA loading
Inhibit DNA binding
Inhibiting DNA cleavage

We performed docking between Cas12g and different mutants of Acr to asses the best results then we settled on Cas12g-AcrIIAv2-Complex which has Docking Score = -268 which is the highest docking score of different mutants of Acr

Due to our concern with the off-targeting events mediated by CRISPR-cas system we carried out computational modeling in order to predict the site where sgRNA would interact with.

There are several methods to control or regulate the activity of Acr-proteins which are divided or acted at several different level one of them is ligand-inducible, so we develop 2 modules of regulation:

1-ligand-inducible Acr protein:
which is developed by fusing AcrIIAv2 to a MS2 aptamer-destabilization domain (DD) (MS2-DD), so. In the presence of TMP, the DD is stabilized, allowing AcrIIAv2 to maintain its structural integrity and repress Cas12g activity. In the absence of TMP, AcrIIAv2 misfolds and becomes degraded, thereby liberating Cas12g. This finding demonstrates the value of using inducible Acr proteins to control Cas proteins that maybe not amenable to direct regulation or further engineering.

2- Conditional gene expression systems Based on CreERT2:
The Cre loxP system can be employed to catalyze recombination in a specific site between its recognition site through Cre recombinase activity. This system can edit genes in different ways, such as deletion, inversion, and translocation between chromosomes;this reorganization depends on the loxP sequence site and position.
If the loxP sequence is in the same direction on the same strand it will lead to the deletion of the gene in between, but if the arrangement is on the opposite side, it will cause reversible inversion of the gene in between. If the same loxP is present on two different strands, it will translocate the upstream gene.
This technology can be applied to induce gene expression by deleting a (STOP) sequence that hinders transcription of the forward gene and this (STOP) is formed of nlacZ with nuclear localization signal, so lacZ is excessively expressed. This (STOP) sequence will be replaced by our reporter gene which expressed during the absence of Cre recombinase

So to get a conditional form of that system in order to work under our control, Cre-recombinase activity is suppressed by adding to it triplet mutant form of human oestrogen receptor, preventing its entry into the nucleus to delete the loxP-flanked (STOP) sequence, but once tamoxifen is available,CreERT2 moves freely into the nucleus, where Cre recombinase activity takes place. and to avoid the side effects of tamoxifen in the previous method, we had to integrate another technology for conditioned gene expression to regulate the activity of Cre recombinase. This novel approach is called Tet-On 3G: inducible system which is a modified technique for gene expression and originated from rTet system, this platform based on two central elements (tTA) the tetracycline (tet)-controlled transactivator that is modified to Tet-On 3G and specific responsive promoter (Ptet) which enhanced to TRE3G promoter Which controls the expression of our transgene (Cre recombinase ), this Tet-On 3G doesn't induce TRE3G promoter until the external element (Tet and tet derivatives) such as doxycycline hydrochloride (Dox) is present, which improves Tet-On 3G DNA-binding capacity thereby performing transcription activation of TRE3G promoter,

And our transgene (Cre recombinase) is freely expressed to target the loxP sequence that is flanked to the (STOP) sequence that hinders the downstream gene (anti-CRISPR) from being expressed, After the deletion of the (STOP) sequence by Cre recombinase activity the transcription of nLacZ will be replaced by anti-CRISPR expression

Our circuit is regulated by the presence of L7Ae-Kt riboswitch that is mediating translational regulation of the expression of the dcas12g. We designed this crispr-cas12g system to cleave the circuit at the site of beta-galactosidase in the absence of the analyte (phe).

This can make certain that there is no chance for the circuit to produce beta-galactosidase(LacZ) if serum-phe levels are not elevated. Hence, the L7Ae/Kt interactions with dcas12g-gRNA can verify the reduction of the false positive results by eliminating the probability of continuous expression of the B-galactosidase in the absence of excess-phe.
The L7Ae-mediated control of the gene expression in our diagnostic circuit represents a modular regulatory element for the crispr expression.
In a similar way this L7Ae/k-turn ON/OFF switch regulating and adjusting the translation of the output protein (dCas12g) is also implemented in the design of our therapeutic circuit. Thereby, this Riboswitch mediated regulation of the crispr system represents feedback regulation for the PAH gene expression in mammalian cells.

This diagnostic platform is considered as an optimum alternative for the traditional tandem mass spectrophotometry. Based on previous literature, results of newborn screening using MS/MS are inaccurate because of false negatives. According to the data provided by the Egyptian national newborn screening program regarding the results of screening practices in Egypt.
We have designed our lateral flow assay to tackle and solve all the potential difficulties of screening tests in terms of: reducing false positives and false negative results. Accordingly we started with determining a cut-off value for the normal level of phenylalanine in the blood sample, which was found to be almost (2-20 mg/dl). Typically, there are many reasons that can cause elevation of phenylalanine in the blood. We have a wide range of causes for serum elevated phe-levels. As the enzyme deficiency doesn't resemble the only cause, however, TetrahydroBiopterin deficiency as a co-factor also affects the levels of the analyte. PKU is a heterogeneous disease, as the method of feeding in newborns was found to greatly affect the phe-levels in the blood. Newborns who are breast-fed showed decreased levels of serum phenylalanine, compared to formula-fed infants. Breast milk contains only 12-14 mg of phenylalanine per ounce but the formula provides 24-28 mg per ounce. This means that diet and feeding methods can greatly affect the results of the screening using MS/MS. Our point-of-care aims at providing a highly sensitive and selective approach through the use of single-stranded DNA aptamers capable of consuming the normal levels of phenylalanine to that cut-off value. This can ensure the reduction of false positive results through a semi-quantitative measure ,through our software tool, of the excess amount left of this analyte that will then be recognized by the help of a whole cell biosensor. This biosensing system depends on an E.coli transformed with our genetic circuit designed to release Beta-galactosidase upon sensing any excess phe that was not consumed by the aptasensor. This genetic system can link the presence of this analyte (phe) to the level of expression of the (Beta-galactosidase) and detection method will be in the form of color change of the paper strip, due to the presence of X-gal pigment upon which the secreted enzyme will act, giving a blue color. This can provide more accurate results in terms of reducing the false positives.
Considering that in aptamer-based LFA, specificity and sensitivity can be affected by the presence of other molecules or analytes with similar structures, leading to false positive results. That’s why we tested the aptamers against various phenylalanine analogues to make sure that the used sequences of aptamers are specific for phenylalanine We also provided a solution for inaccurate results that may result from non-viability of the cell-based biosensor. So, we added a control line of an E.coli based system capable of expressing LacZ-alpha on a regular basis. This control line is an example of the validation steps that can guarantee the stability, viability and proper expression function of the used e.coli biosensor for the phenylalanine analyte)

Our team has given more care to the biosafety and security aspects of the project in order to ensure that our designs are safe and can be safely regulated by the CRISPR-Cas system. In addition, we've come up with another approach to add more safety layers to the system in the form of what is called a Riboswtich, which is an RNA-binding protein that can control the genetic circuit depending on the concentration of the biomarker of interest. We are also creating a novel and universal platform for regulating all the designs by making on-off switches if the circuits get out of control. Hence, to make sure that we are working tirelessly on various safety modalities and testing them at the same time.

Our team provided a Biosafety course for the future iGEM teams, to help and guide them during their lab-phase.