Table 1 In vivo genotype diversification strategies for in vivo continuous evolution

Modified natural mutagenesis system

PolIII

No

N/A

≈ 10⁻¹⁰

No

Introducing error-prone dnaQ into the system to increase mutation rate

[23]

PolI

Yes

650 bp

≈ 8.14 × 10⁻⁴

No

Targeted gene is transferred to the preferential region of low fidelity variant of PolI to achieve error-prone transcription. The mutation rate can be enhanced by the absence of mutL and mutS system. However, this system is distance dependence

[27]

Plasmid-targeted mutagenesis system

OrthoRep

Yes

3.7 kbp

≈ 10⁻⁵

No

Orthogonal DNA plasmid-DNA polymerase pair extranuclear replication system is used. Another TP-plasmid containing targeted gene is prepared to be targeted by ep-DNAP to increase the mutation rate on the targeted region while maintaining the nature mutation rate of the plasmid containing all the essential genes

[36,37,38]

Genome-targeted mutagenesis system

TaGTEAM

Yes

20 kbp

≈ 10⁻⁷

No

Targeting the binding site of DNA glycosylase (MAG1) and DNA binding protein (tetR) with a mutation generation system through ep-HR by resectioning and ep-Pol ζ

[39]

EvolvR

Yes

350 bp

≈ 10⁻⁵–10⁻⁶

Yes

Point targeting of targeted gene using CRISPR-nCas9, and mutate the targeted gene with DNAP Pol3 M attached to CRISPR-nCas9

[41]

Base editing

Yes

18–23 bp

(100 bp for CRISPR-X)

N/A

Yes

Point targeting of targeted gene using Cas9 protein, while editing base using base editor attached to Cas9 protein

[42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59]

Recombination-based Mutagenesis System

SCRaMbLE

Yes

N/A

loxPsym dependence

Yes

Insertion of loxPsym after the stop codon of non-essential gene, and trigger chromosomal rearrangement using Cre recombinase

[63, 65,66,67,68,69]

Retron

Yes

5 kbp

≈ 6.67 × 10⁻³

Yes

The targeted gene labelled with retrotransposon recognition label is transcribed, followed by reversed transcription to generate specific mutation on gene

[70, 72, 73]