Stanford iGEM Team Wiki

Novel Directed Evolution Application


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This video details the DiCE in vivo genetic circuit we designed to evolve a novel anti-CRISPR, AcrIIA4. We recommend first reading the Description section for project motivation and background!


In the last 5 years, upward of 450 thousand genetically modified (GM) mosquitoes were released in Jacobina, Bahia, and Brazil [2]. These mosquitoes carried a powerful and deadly gene drive system that has a synthetic dominant inheritance pattern designed to wipe out large portions of the mosquito population in the cities where they are released. With a huge potential for these GM mosquitoes to help control malaria and other blood-borne diseases around the world, these gene drive technologies are rapidly being developed in labs across the country, with mass implementation on the horizon.

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Promise and peril: Genetically modified mosquitoes released in Brazil.

However, in an ominous and recent publication, genetic evidence emerged that the genetic modifications of the engineered species has spread to mosquitoes beyond the intended target population [3]. Indeed, since the technologies inception environmental experts have warned that "this is an experimental technology which could have devastating impacts [4]," and this recent publication with unexpected genetic consequences is one of the first examples of the peril of this technologies.

This is where AcrIIA4 comes in—anti-CRISPRs have the potential to provide an “off-switch” to gene drives (see Background below), and thus give scientists a safety net when releasing organisms with this lethal technology. To safe-guard against aberrant and unintended consequences of “gene-drives gone wrong,” our team aimed to develop a novel anti-CRISPR that could provide an “off-switch” to xCas9-based gene drives. Thus, we hope to further the powerful potential of these disease-eradicating gene drives with a foundational advance that could increase safety.


The CRISPR-Cas9 system presents exciting possibilities in the fields of bioengineering and synthetic biology, due to its DNA specificity and versatility. It has functioned as the cornerstone for several previous iGEM projects, from site-directed mutagenesis to DNA detection systems.

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Figure 1: NGG PAM necessity: This diagram depicts Cas9 (grey) being guided by sgRNA (multi-colored) to a PAM-adjacent recognition sequence (PAM=yellow, Rec. Seq.= orange) on the target DNA (blue)

However, Cas9’s gene-editing applications are limited by its dependency on a specific protospacer adjacent motif (PAM) upstream of the targeted nucleotide sequence, the least restrictive of which was formerly NGG. However in 2018, Hu et al. used phage-assisted continuous evolution (PACE) to evolve a Cas9 variant (xCas9) with a less specific PAM requirement [5]. Given its increased effectiveness and versatility, this novel Cas9 variant has continued to grow in popularity.

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Figure 2: Anti-CRISPRs block Cas9 from binding to DNA. This diagram shows our the goal of this subproject: designing the anti-CRISPR AcrIIA4 to block dxCas9 gene knockdown.

This new xCas9, however, has not been shown to have anti-CRISPR proteins that can effectively deactivate it. Anti-CRISPRs are small proteins that can block the Cas9 protein from binding to DNA and thereby prevent genetic modification or downregulation typically caused by Cas9. Anti-CRISPRs are important for designing novel genetic circuits involving CRISPR, and most importantly for our project, they provide a safety net for CRISPR-based gene drives.

While we will not get into the genetic details of CRISPR-based gene drives (we recommend that article though!), Cas9 is the critical protein that leads to the engineered dominant inheritance pattern. What anti-CRISPRs allow is a way to block the Cas9 and thereby shutdown the gene drive. Hence, when designing a genetic circuit for a CRISPR-based gene drive, scientists can put in an anti-CRISPR under an inducible promoter as a countermeasure to activate if a gene drive does not function as expected [6].

Thus, as xCas9 becomes more popular and a likely candidate for future gene drives, it is imperative that we develop an anti-CRISPR that can effectively shutdown this gene drive if something goes wrong.

Since xCas9 is similar in structure and sequence to Cas9, and AcrIIA4 is the anti-CRISPR associated with Cas9 deactivation, we decided AcrIIA4 would be a good starting point for our DiCE evolution in order to eventually evolve a novel anti-CRISPR that could block xCas9.


For our first step towards our goal of designing AcrIIA4 to be more effective at inhibiting xCas9, we had to determine if AcrIIA4 already has some activity against xCas9. Based on our literature search, AcrIIA4 has never been tested against xCas9. There are ten amino acid differences between Cas9 and xCas9. However, two occurred in the AcrIIA4 binding site, and the other eight could have also potentially resulted in conformational changes to the protein that could affect AcrIIA4’s ability to interact with the xCas9 (see model). Based on this modeling, we suspect that AcrIIA4 may not be effective against xCas9 to the same degree as it was with Cas9.

Video Caption: This model supports our hypothesis that AcrIIA4 likely does not effectively inhibit xCas9. Specifically, it shows Cas9 (blue) in its activated form bound to sgRNA (white) and AcrIIA4 (yellow). The AA changes from Cas9 to xCas9 (of which there are 10) are highlighted in green. Cas9 AAs that are within 5 Aº of the AcrIIA4 are highlighted in red, while AAs in the 5-10 Aº range are highlighted orange. Of the 10 xCas9 AA change, two were in the binding pocket of AcrIIA4 (one within 5 Aº and one between 5 and 10 Aº). These two AA changes could likely interfere with AcrIIA4’s ability to interact with xCas9.

This modeling data justified our next steps: designing a system to test AcrIIA4’s current effectiveness against xCas9. We took two approaches to answer this initial question:

1) In Vitro Construct Design
We first designed a cell-free system to test the efficacy of AcrIIA4 against dxCas9, due to its time-effectiveness. Cell-free reactions involve simply the gene fragments of interest and basic cell machinery. Since each linear fragment can be put into the system independently, there are no plasmid constraints typical of in vivo work, resulting in less cloning work. We obtained dCas9, AcrIIA4, and two sgRNA constructs from the Stanley Qi Lab. The two sgRNA constructs consisted of an “Scr sgRNA” that had a scrambled variable targeting region (negative control), and an “RR1 sgRNA” that could guide dCas9 to the RFP gene and thereby silence it. We designed primers that bound to the start and stop codons of these genes, and then added overhangs with a T7 promoter, RBS, and T7 terminator. Additionally, in this cloning process we created a “dead” version of AcrIIA4 that had a frameshift mutation leading to a premature stop codon. Finally, we used a standard RFP plasmid from our lab that already had a T7 promoter and terminator. This RFP was our target gene for the dCas9 and dxCas9, and could visually indicate knockdown status as well as produce data on our plate reader.

An abridged version of our AcrIIA4 cell free linear construct in Benchling. The primers we used had heads (highlighted in brown) that bound to the AcrIIA4 gene sequence in the plasmid obtained from the Stanley Qi lab. The overhangs for these primers added the necessary promoter (green), RBS (blue) and terminator (grey) needed for cell free expression with just one simple PCR.

2) In Vivo Construct Design
In addition to pursuing AcrIIA4 testing in cell free, we also wanted to design DNA constructs to test its activity in vivo. For this we ran into the problem of needing to put our four different components (1. dxCas9/dCas9 2. RR1/Scr sgRNA, 3. AcrIIA4/dead AcrIIA4, and 4. RFP) into a three plasmid system. Given the starting plasmids we had to work with, the constraints of origins of replication and antibiotics, and aiming to minimize the number of Gibson Assemblies required, we came up with the following cloning plan.

Our in vivo cloning strategy to test AcrIIA4’s activity against dCas9 (control) and dxCas9 (test).

Unfortunately, while we successfully cloned AcrIIA4 onto the sgRNA plasmid and sequence verified the result, we struggled with the backbone PCR to swap out the dCas9 for the dxCas9. At this point, due to initial promising results from the cell free testing, we decided to optimize our time by halting the in vivo cloning and focusing on testing in vitro.


Our first experiment was to test AcrIIA4 against both dCas9 and dxCas9 in cell free. We devised the following test groups:


We used the PURExpress® In Vitro Protein Synthesis Kit from NEB and ran our test groups in duplicates for 5 hours. During this time, we monitored fluorescence every ten minutes in our plate reader with excitation set to 520 nm and emission measurement at 610 nm.


Because cell free is expensive, we started with testing just the dCas9 groups first to demonstrate that all of our individual components were functioning as expected. Following a few suboptimal trials where we struggled with bubbles in the plate reader, we obtained the following data.


Our positive and negative controls gave the expected results: our RFP fragment fluoresced, and our H2O negative control had minimal background. We saw marked lower expression for one our RFP positive controls, perhaps due to pipetting error.

Our RFP + scrambled sgRNA + dCas9 group fluoresced as expected (because the sgRNA variable region is scrambled and therefore unable to guide the dCas9 to the RFP gene for knockdown). What’s more, our trial with RR1 and dCas9 led to significant decrease. However, these initial results did not demonstrate AcrIIA4-dependent return of RFP expression: both our RFP+RR1+dCas9 + either dead AcrIIA4 or normal AcrIIA4 groups behaved the same and did not fluoresce.

After rerunning these AcrIIA4 groups specifically and seeing similar results, we sequence verified to confirm that we had successfully added the T7 promoter, RBS, and T7 terminator to our linear fragment. Because this sequencing data checked out, we were left with inconclusive results for AcrIIA4. We had two hypotheses as to why this might have been:

    In Vitro
  1. Occasionally proteins that are functional in vivo are not able to fold properly in cell free extract. Though this is uncommon it is possible that AcrIIA4 might have been one of these proteins.
  2. Perhaps more likely, the AcrIIA4 was struggling to express because it’s DNA sequence was mammalian codon optimized instead of bacterial. Though it is often not a problem to express mammalian optimized vectors in bacteria or bacteria extract, it can sometimes lead to far lower expression levels that could be responsible for the observed lack of effectiveness.

At this point in time, we unfortunately realized we were running low on our PURExpress kit. The cost to purchase more reactions was significantly high and we were still quite far from answering our initial question, so we decided to pivot our energy to other subprojects.

Instead, we shifted our focus to implementing a cost-effective protocol to develop our own cell-free extract, which we called ourTXTL. Check out this process here! Our hope was to return to cell free testing once we finalized this protocol, but unfortunately we only got successful results with ourTXTL later in the summer at which point it was too late to pick up this project again!

We did, however, carefully design a three plasmid system that could effectively evolve AcrIIA4 to be more effective against dxCas9 using the DiCE in vivo evolutionary system. See below.


While we were not able to conclude whether AcrIIA4 is effective against dxCas9 this summer, we do indeed think this project is worth pursuing in the future. Especially given that cell free expression is far more accessible given the ourTXTL process we worked on this summer, we hope to return to additional testing following the Jamboree.

Furthermore, we went ahead and envisioned the following DiCE in vivo selection schema to evolve AcrIIA4 to be better at inhibiting dxCas9. Once we get the answer to our initial question of AcrIIA4’s current effectiveness against dxCas9, we can pretty easily clone the following system by simply swapping in AcrIIA4 to be between the Qbeta binding MDVs with restriction enzymes, and changing the 20 base pair variable region of the sgRNA to block CmR expression. See our video for a more detailed explanation of our AcrIIA4 DiCE selection system!