Targeted Mutagenesis with EvolvR

Mutagenesis is a tool that both evolution and molecular biologists use to tinker with DNA. Making changes to a DNA sequence can help scientists identify and/or facilitate the evolution of new phenotypes, and forward genetics harnesses this at a large scale by screening diverse libraries of genetic variants. Several methods for generating mutant libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants in bacteria. Read on to learn about the key components of EvolvR and its potential applications.

Challenges of existing mutagenesis methods

Several continuous diversification methods exist, but there are a few drawbacks with these approaches that makes it challenging for users to achieve targeted diversification of all nucleotides within a defined region of a genome.

• Require stringent bacterial growth conditions. Expression of an error prone polymerase I (PolI3M) is capable of mutagenesis in E. Coli, but this approach requires special bacterial growth conditions in order to maximize the mutation rate. For example the Phage Assisted Continuous Evolution (PACE) method requires constant turbidity of the culture, so bacteria must be grown in a turbidostat.

• Only mutate particular types of nucleotides. Both the Bassik Lab and the Chang Lab have created methods that introduce diversification by targeting a site of interest with dCas9 fused to the Activation Induced Cytidine Deaminase (AID) enzyme. AID, however, only changes cytidines or guanines to the other three bases, which limits the level of variation that can be introduced.  

• Diversification is limited due to the integration of oligonucleotide libraries of a discrete size at the target site. The use of such libraries means that the diversity introduced by lambda red recombineering and Multiplex Automated Genome Engineering (MAGE) will always be limited by the size of the library used.

EvolvR: key components

The EvolvR system has two key components: the error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar to mutagenesis with error prone PCR, EvolvR uses an error prone polymerase to introduce mutations. However, by using nCas9 to direct PolI3m to a particular genomic loci, EvolvR allows for targeted mutagenesis of a site of interest. A gRNA is used to direct the PolI3M-nCas9 complex to a DNA site of interest, which nCas9 nicks and then dissociates from. PolI3M then binds the nicked DNA, and extends it from the 3’ end, while its native endonuclease activity degrades the displaced strand.  

Figure 1: EvolvR consists of an error-prone polymerase (PolI) that is fused to a nicking Cas9. A guide directs Cas9 to a site of interest, where it nicks its target DNA (left). Cas9 dissociates and PolI binds (middle) and extends from the 3' end of the nick (right), occasionally introducing mutations (red nucleotide). Created with BioRender.com.

The initial version of EvolvR has an editing window of ~17 nt from the nick site, which was expected since the processivity window of PolI is 15-20 bp. EvolvR’s on-target mutagenesis rate was 2.5 x 10^-6 mutations per nucleotide per generation vs. 10 x 10^-10 mutations per nucleotide per generation of wild-type E. coli, while only increasing the standard mutation rate seen during DNA replication by 120-fold over background.

Due to its modular nature, the Dueber Lab created a few versions of EvolvR. To increase the mutation rate of EvolvR, three additional amino acid changes were introduced to nCas9. These changes promote nCas9’s dissociation from DNA after nicking and yielded an enhance nCas9 (enCas9) EvolvR with a targeted mutagenesis rate ~9-fold higher than the original nCas9, while increasing the standard mutation rate by only ~2-fold over nCas9 levels. Two sets of modifications were also made to PolI3M to increase the mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M generated PolI5M which increased EvolvR’s mutagenesis rate to ~10^-3 mutations per nucleotide per generation, and (2) the addition of the thioredoxin-binding domain (TBD) from bacteriophage T7 DNA polymerase increased the processivity of PolI3M, and thereby increasing the editing window of EvolvR to ~56 bp. A larger editing window also increased the likelihood that a single gRNA would introduce more than one mutation near the target site.

Applications of EvolvR

The Dueber Lab used EvolvR to discover novel genotypes that result in a spectinomycin resistant phenotype in bacteria. While there are many known spectinomycin resistance mutations, several new mutations were identified by targeting EvolvR to five dispersed regions of the target gene of spectinomycin, rpsE. These results not only uncovered novel resistance genotypes, but also provide a better understanding of the key residues responsible for this protein-drug interaction.  

The Dueber Lab propose that EvolvR could also be used to map protein-protein interactions, investigate non-coding segments of the genome, or engineer microbes to perform tasks.

EvolvR could also be a tool for lineage tracing cells that do not tolerate double-strand DNA breaks by introducing a unique tag into a genomic site of choice via the mutational abilities of PolI3m. This tag would allow researchers to trace the progeny of a single starting cell. Additionally, multiplexing of guides allows EvolvR to target more than one genomic site for mutagenesis, a feature which they used to generate bacteria resistant to both spectinomycin and streptomycin, but could also be used for studying epistatic interactions.

Key takeaways for editing with EvolvR

EvolvR allows for the continuous diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in defining the mutational rate and editing window of the system and the flexibility to address many different types of research questions. Are you ready to start EvolvR-ing your targeted mutagenesis experiments? Find EvolvR plasmids at Addgene!

---

References

Gaj, Thomas, and Pablo Perez-Pinera. "The continuously evolving CRISPR barcoding toolbox." Genome biology 19.1 (2018): 143. PubMed PMID: 30253789. PubMed Central PMCID: PMC6154929.

Halperin, Shakked O., et al. "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window." Nature 560.7717 (2018): 248. PubMed PMID: 30069054.

Sadanand, Saheli. "EvolvR-ing to targeted mutagenesis." Nature biotechnology 36.9 (2018): 819.

Additional resources on the Addgene blog

• Learn about other CRISPR methods for bacterial genome engineering  
• Learn more about lambda red recombineering of bacteria
• Use site directed mutagenesis to generate specific mutations in your plasmids
• Use REPLACER Mutagenesis to mutate your plasmid

Resources on Addgene.org

• Browse our CRISPR guide pages
• Find bacterial CRISPR plasmids