See also CRISP gene editing under Enzymes and endonucleases

Gene editing refers to the introduction of desired changes at specific genomic loci. The original strategy for gene editing was to use programmable sequence specific nucleases to generate NA double strand breaks at predetermined gehomic sites, the desired changes being produced by subsequent non-homologous end joining, microhomology mediated end joining or homology directed repair. The emergence of CRISPR-Cas systems accelerated the development of gene editing technologies. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022). 

CRISPR

CRISPR based methods creat edits by using a guid RNA to efect targeted breakage, and a plasmid-borne donor DNA to repair these breaks with the desired sequence, whith phenotype tracking permitted by amplicon sequence of these components. 

Retron Library Recombineering (RLR)

Retrons are prokaryotic retroelements that that under targeted reverse transcription, producing single stranded multicopy satellite DNA (msDNA). They participate in anti-Phage defense mechanisms. These components can produce functioning recombineering donors within cells, creating specified genomic cells. Pooled, barcoded mutant libraries can be prepared in this way and used for characterization of natural and synthetic allelic variants, a process referred to as RLR. RLR is an alternative to CRISPR based methods above and has the advantage that the rLRs “donor only” versus CRISPR’s “guid + donor” method eliminates the requirement for ablating CRISPR targetting, allowing single based pair changes to be characterized without wihtout requring additional prtospacer adjacent motif (PAM) targetting. This allows ablating mutations to be incorporated. RLR overcomes the requriement for targetting a suitable PAM altogether, whereas CRISPS “guide + donor” methods decrease in performance as the distance to a PAM increases. RLR’s lack of guide simplifies RLR elements. In contrast to the two unique elements required for the “guide + donor” strategies, RLP’s solle requirement is a unique short donor  sequence within the retron. This relaxed design constraint enables RLP using nondesigned variation. (Church, PNAS 2021, 118 (18) 

Eukaryotic retroelement Proteins for transgene insertion:

(Zhang, “Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci, 2024) discloses precise RNA mediated insertion of transgenes (PRINT), an approach for site specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro trasncribed RNAs: messenger RNA encoding avian R2 retroelement protein and template RNA encoding a transgene of lenght validated up to 4 kb. The R2 protein cocordinately recognizes the target stie, nicks one strand at a prcise location and primes complementary DNA syntehsis for stable transgene insertion.