“Evolution is change in the heritable characteristics of biological populations over successive generations.”
——Brian K. Hall

Evolution is a slow process that continually occurs in nature while it can't achieve the optimal quality as expectation for industry production. This stimulates researchers to find a new way to make the evolution process more compatible with favorable outcomes for humans.

Then comes directed evolution, a groundbreaking innovation. With directed evolution, we can get optimized proteins through cycles of diverse library construction and selection. In other words, directed evolution is an ideal optimization system of functional proteins.

We choose Cas9 protein as our candidate. CRISPR-Cas9 system is an efficient gene editing method. However, its off-target problem has been troubling researchers a lot. Regarding this, our project decides to conduct optimization methods to obtain a high fidelity CRISPR-Cas9 system for specific target. Therefore, we get LuCas9, Locational Unique Cas9. In our LuCas9, the off-target effect would no longer be a troublesome factor.

Figure 1: Diagram of the direction of directed evolution.

Rational Design

Off-target prediction model can detect and select off-target sites from genome. We use "Lure" (off-target) sequences and "Target" (on-target) sequence predicted by this model to construct our screening system.

The SpCas9 mutant library is built by error-prone PCR. We use the CRISPRi (inhibition) system and fluorescent proteins to orthogonalize the on/off target effect into red and green fluorescence. Once the dCas9 protein binds to the target/lure sites, the expression of downstream proteins will be suspended.

With this reporting system, we use FACS (Fluorescence activated Cell Sorting) to screen eligible strains. After cycles of mutation, selection and shuffling, advantageous mutations are accumulated, eventually achieving the effective directed evolution and obtaining our dream protein.

You can read more about our project design here.


After the screening process, we need to analyze the efficiency of directed evolution system. Our kinetics model can evaluate the change of free energy during the targeting progression. Therefore, we can infer the potential energy of LuCas9 binding process.

You can read more about our kinetics model here.


Although directed evolution is theoretically capable of achieving the "optimal solution", there are still some defects. For example, we need to start a new evolution process even if we only want to make small changes to known features.

How can we further improve the process?

Rational design can obtain the evolutionary prediction results calculated by its model. Thus it is an ideal method to assist our directed evolution progress. We first discovered the difference between the distances of the key residues of SpCas9 and xCas9 and the DNA-sgRNA double helix. With the help of molecular dynamics, we can predict the mutant structures, and then their off-target effects.

Furthermore, with rational design, we can customize proteins with more specific demands. For example, we can construct Cas9 proteins which have high tolerance for mismatches in specific locations.

You can read more about rational design here.

Figure 2: The relationship between directed evolution and rational design.


Our project provides all researchers with a method to obtain high fidelity Cas9/dCas9 protein. This can benefit labs and companies which want to implement gene editing for specific target.

In addition, we conducted a survey on public attitudes towards gene editing technology to understand people's opinions about it.