New Technique Could Challenge CRISPR by Editing Millions of DNA Sites Simultaneously

Gene editing has enabled us to push the limits of synthetic biology with multiple technologies such as CRISPR-Cas9, TALENs (transcription activator-like effector nucleases), and ZFNs (zinc-finger nucleases), which focus on cutting the DNA sequence at specific sites to edit it.

While these have been highly versatile and improvised for multiple applications, they are limited in scale. Using recombination and a retron library, scientists from the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School have designed a high-throughput gene editing method called Retron Library Recombineering (RLR).

RLR can edit millions of DNA sites simultaneously, where each mutation can be followed and screened with its unique barcode. This enables simultaneous and quantitative analysis of millions of experiments which can help understand the interplay of mutations at the whole genome level.


Retron Library Recombineering

Like CRISPRs, retrons were found to have evolved to function as antiviral defense systems. Although discovered many decades earlier, their anti-phage function was uncovered only recently. Retrons are unique bacterial DNA sequences that are a complex of DNA, RNA, and protein components. It exists as a multicopy single-stranded DNA (msDNA) that undergoes reverse transcription to form single-stranded satellite DNA.

In this study, published in the May 2021 issue of the Proceedings of the National Academy of Sciences, the retron was engineered to contain a mutation. A library of retrons containing a wide array of mutations was introduced into a bacterial population along with the enzyme reverse transcriptase to produce ssDNA, which was incorporated into the replicating bacterial genome by recombination.

The retron sequences served as barcodes and were used for quick mutant screening in a population of bacterial cells containing multiple mutations. Instead of forcing mutant DNA into the cells, retrons allowed for the synthesis of mutant DNA sequences inside the cell without damaging the bacterial DNA. The mutation in the ssDNA was incorporated into the bacterial genetic code in subsequent rounds of replication by the single-stranded annealing protein (SSAP). The resulting mutants were screened for their phenotype.

Retron sequences (red) containing a mutation of interest (black notch) are introduced into a bacterial cell along with the enzyme reverse transcriptase (RT). The retron then produces ssDNA that is inserted into replicating DNA with the help of another enzyme called single-stranded annealing protein (SSAP). Image Credit: Max Schubert / Wyss Institute at Harvard University

The initial prototype design was to introduce antibiotic resistance mutations in the retrons so that they could be easily screened. The RLR technology was sensitive to pick up subtle differences among mutations that varied in their antibiotic resistance response.

In a step-up, the authors fragmented antibiotic resistant E. coli genome and built a multi-million sequence retron library to edit an RLR-optimized bacterial strain. Editing efficiency was improved by deleting exonucleases and inactivating the mismatch repair module, which could clear the ssDNA in the cell.


Advantages Over CRISPR

A distinguishing feature of RLR is that the cell population containing the mutation is enriched by multiple rounds of bacterial replication, which enhances its efficiency. It could be particularly helpful in cells where CRISPR is not ideal for gene editing due to toxicity or high off-target effects. 

In a press release from the Wyss Institute, co-first author Max Schubert, Ph.D., a postdoc in the lab of Prof. George Church, said, “RLR enabled us to do something that’s impossible to do with CRISPR: we randomly chopped up a bacterial genome, turned those genetic fragments into single-stranded DNA in situ, and used them to screen millions of sequences simultaneously.

“RLR is a simpler, more flexible gene editing tool that can be used for highly multiplexed experiments, which eliminates the toxicity often observed with CRISPR and improves researchers’ ability to explore mutations at the genome level,” said Schubert.

Besides, retrons enable the construction of a much bigger library than what is currently used with CRISPR. This is because retrons can edit genomes with only a donor DNA, while CRISPR requires both a guide and a donor DNA sequence to induce each mutation.

RLR could vastly enhance the scale and flexibility of genome editing and can also be coupled with CRISPR-based experiments. The authors recognize that replication-dependent editing may not be suitable for all cell types, and more work is needed to increase editing efficiency, decrease allele-specific efficiency, and to evaluate if RLR is viable for alterations such as inversions and duplications.  Nonetheless, RLR is a promising high-throughput tool to study multiple mutations and their interactions in the genomic context.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: The Game Changer: New CRISPR Tool Can Turn On/Off Genes

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