Fig. 7 |. Retrons allow recombineering without exogenously delivered DNA.

a | Synthetic cellular recorder integrating biological events (SCRIBE) uses retrons to produce multicopy satellite DNA (msDNA) in vivo, which is used as a substrate for recombineering. The presence of an induction molecule triggers expression of a reverse transcriptase (RT; orange) and a retron transcript (msr/msd). The msd portion of the retron transcript is reverse transcribed by the RT to create msDNA. This msd region can be modified to include a customized loop that carries a user-defined mutant allele. A single-stranded DNA-annealing protein (SSAP) then mediates incorporation of the msDNA into the nascent copy of the genome at the lagging strand of the replication fork. b | Error-prone T7 RNA polymerase (RNAP) can be used to produce mutagenized msr-msd transcripts. These transcripts are reverse transcribed to produce a library of msDNA editing templates that contain random mutations for continuous evolution of a target sequence. c | Retron library recombineering is a technique for genome-scale reverse genetics. Synthetic or natural DNA variation is encoded into the retron msd element on a plasmid library. This plasmid library is transferred to a population of bacteria, the retron directs a mutation into the genome and the population is taken through a screen or selection. The prevalence of each allele within a population can be tracked by sequencing of a plasmid amplicon containing the retron msd. NSG, next-generation sequencing; ssDNA, single-stranded DNA.