Retron Library Recombineering: A New Solution for Genome Editing after CRISPR/Cas    

The biotechnology sector is reaching a tipping point, with decreased prices and greater speed of DNA synthesis and sequencing making it possible to change particular base pairs in genomes. Compared to previous technologies such as meganucleases, zinc fingers, TALENs, and CRISPR/Cas systems, Retron Library Recombineering (RLR) offers potential for future innovation. The CRISPR/Cas system generates desired alterations by targeting and cutting certain DNA segments; however, the Cas enzyme may cut non-target locations, complicating the process and potentially injuring cells.

The Mysterious Nature of Retrons

Retrons are DNA fragments found in bacterial genomes, typically about 2kb in size. Recent studies have shown that retrons encode reverse transcriptase (RT) and non-coding RNA (ncRNA). The msr and msd regions are usually present within retron ncRNA, folding into specific secondary structures. During translation, RT binds the RNA template and starts reverse transcription, generating single-stranded DNA/RNA hybrid molecules that play a crucial role in genome editing. Numerous studies have revealed the role of retrons in bacterial immune systems, making them an attractive biotechnological tool.

Principles and Mechanisms of Retron Library Recombineering (RLR)

Retrons are genetic elements within bacterial genomes that synthesize single-stranded DNA (ssDNA), offering new possibilities for genome editing. The basic process of RLR technology involves:

• ssDNA Synthesis: Utilizing retron elements in bacterial genes to synthesize ssDNA containing target mutations in vivo.
• Genome Integration: These ssDNA molecules serve as templates during the DNA replication process (especially lagging-strand synthesis) and are directly introduced into the genome through homologous recombination. This process does not require DNA cutting or complex repair mechanisms like CRISPR.
• Multiple Mutations: RLR can introduce multiple mutations simultaneously, using barcodes for high-throughput screening and analysis of mutants.

Figure 1. Overview of retron-mediated genome editing in eukaryotic cells. (Kaur N, et al., 2024)

Prokaryotic Genome Editing Based on Retrons

RLR has proven to be a multifunctional and versatile genome editing tool, superior to other marker-less editing methods such as oligonucleotide recombination. By transcribing retron msr/msd regions and reverse transcription, multi-copy satellite mutant DNA generated within cells can serve as templates to introduce desired variations during replication. Recent studies have enhanced the performance of RLR by disabling inherent mismatch repair systems in cells to increase mutation success rates and extend its application range in prokaryotes.

Combined CRISPR/Cas Genome Editing in Eukaryotes

In eukaryotes, the retron system is used in combination with CRISPR/Cas for genome editing. In yeast cells, an improved retron mechanism combined with SpCas9 gRNA has achieved precise genome editing through enhanced HDR mechanisms. In HEK293T human cells, retrons from different sources have shown potential for RT-DNA production and significantly increased CRISPR-based HDR events. Additionally, combining the Agrobacterium tumifaciens VirE2 gene has significantly boosted ssDNA production and HDR efficiency in plants.

Advantages of RLR over CRISPR/Cas

Unlike traditional CRISPR methods that require PAMs and a "guide + donor" strategy, RLR uses endogenously generated ssDNA templates to guide mutations, avoiding damage to native DNA. Furthermore, the barcoding feature of RLR allows easy screening of mutant strains via high-throughput sequencing, enhancing experimental convenience and efficiency. In various biological systems, RLR serves as an alternative to CRISPR/Cas or could improve its editing capabilities.

Applications of Retron Library Recombineering

Though RLR has only recently emerged, it has already shown remarkable performance in bacterial genome editing. In prokaryotic systems, retrons have been employed as powerful DNA factories and in vivo genome editing tools. Researchers have successfully optimized retron expression systems and editing conditions, achieving 100% editing efficiency in bacteria such as E. coli.

In eukaryotic systems, retrons have also shown potential for use with CRISPR/Cas. In brewing yeast and human HEK293T cells, researchers have enhanced target site repair efficiency by modifying retron ncRNA and guide RNA constructs, demonstrating promising prospects for retrons in improving the accuracy and efficiency of genome editing. For example, in tobacco plants, using plant viral vectors and Agrobacterium VirE2 protein to co-express an improved retron system and CRISPR/Cas9 has effectively increased HDR efficiency in genome editing.

Creative Biogene—Innovation Drives Excellence

With 15 years of experience in genome editing, Creative Biogene offers global assistance for cutting-edge gene editing technologies. Although RLR, as a nascent genome editing method, requires further research and optimization for eukaryotic applications, Creative Biogene's CRISPR PlatformCB is poised to pioneer this breakthrough technology. Through ongoing innovation and optimization, we are committed to providing cutting-edge technology and comprehensive services for your gene editing projects.

• Exceptional Innovation: We aim to seamlessly integrate Retron and classical CRISPR/Cas technologies to enhance HDR (homologous recombination) efficiency, offering unprecedented flexibility and precision for genome editing projects.
• Rich Experience: Our expert team has accumulated experience in optimizing retron systems and combining them with CRISPR/Cas, providing professional editing services for both eukaryotic and prokaryotic organisms.
• Comprehensive Support: We offer not only advanced RLR technology support but also complete project planning, experimental design, data analysis, and follow-up support to ensure breakthrough progress in your research.

Choose Creative Biogene and explore the limitless possibilities of genome editing. Contact us to jointly achieve your aspiration for precise gene editing!