2.2 Canavanine plates selecting for CAN1 mutant colonies
The mutation rate of CAN1 (with PT7 targeting
sequence) was determined to characterize the efficiency of our
mutagenesis system in this work. Cells were grown to saturation for 24 h
in liquid SC medium lacking appropriate amino acids depending on the
autotrophic markers to maintain plasmids. Cultures were then diluted and
adjusted to OD600=1 and added into inducing media.
Inducing media contains 0.2% galactose and 1 uM β-estradiol. After
incubation at 30℃ for 8~24 h, 30~50 μL
of the cultures were plated onto SC-Leu-His-Arg plates with 60mg/L
canavanine, and the same volume of culture was gradient diluted and
plated onto YPD plates. Colonies on canavanine and YPD plates were
counted after 2~3 days to determine the mutation rate.
Mutation rate analysis
The SC-Leu-His-Arg with 60mg/L canavanine plates were incubated at 30℃
for 2~3 days and the colonies were counted. The sample
size was based on the number of countable colonies on a single plate (up
to 500), and the number of colonies on the YPD plates was used as a
control. Statistical analysis was performed using Graphpad Prism.
Mutation diversity analysis
For mutation diversity analysis, the average-sized colonies were
randomly selected and the target locus was PCR amplified. The PCR
products were analyzed by Sanger sequencing and compared with the
reference sequence.
Results
PmCDA1 increased the mutation rate of the target gene
Cytidine deaminase PmCDA1 can catalyze the deamination of cytosines,
mutating cytosine (C) to uracil (U), while uracil pairs with adenine (A)
in the subsequent DNA repair and replication process to complete the C→T
conversion.[28, 29] Uracil glycosylase inhibitor
(UGI) is usually used to block the activity of uracil glycosylase (UNG)
and inhibit the removal of mismatched uracil, thereby reducing other
types of mutations such as C→G and C→A.[30, 31] We
hypothesized that in the absence of UGI, the types of base substitutions
generated by PmCDA1 might be more diverse.[32] We
constructed the mutagenesis plasmids carrying the pGAL-PmCDA1-T7 RNAP
expression cassette. Meanwhile, we inserted the T7 promoter sequence
upstream of the target CAN1 gene so that PmCDA1-T7 RNAP could be
specifically recruited to the target site defined by the T7 promoter
(Fig. 1A). The mutation rate was characterized by the frequency ofCAN1 gene inactivation. We performed the assay on yeast strains
with and without the mutagenesis plasmids. After induction with
galactose for 8~24 h, we plated cells on canavanine
plates capable of inhibiting CAN1 + cell growth,
and counted colonies on canavanine plates to assess the mutation rates.
Compared with the control strain, the mutation frequency of theCAN1 gene in the strain expressing PmCDA1-T7 RNAP was
significantly increased (Fig. 1C), indicating that PmCDA1-T7 RNAP can
effectively increase the mutation rate of the target gene in S.
cerevisiae .
Appropriate extension of the linker length can sometimes expand the
targeting scope.[30, 33] Based on this, we further
investigated the influence of different linker lengths on the mutation
effect of PmCDA1-T7 RNAP. Two linker lengths (32a.a. and 84a.a.) were
selected (Fig. 1B), and the mutation rate of CAN1 gene was
determined. We observed that the length of linker between PmCDA1 and T7
RNAP had no significant effect on the mutagenic activity of the fusion
protein. After 24 h of induction, both PmCDA1-32a.a.-T7 RNAP and
PmCDA1-84a.a.-T7 RNAP could increase the mutation frequency up to
1.0x10-3~1.2x10-3(Fig. 1D). We analyzed the mutations generated by PmCDA1-32a.a.-T7 RNAP
and PmCDA1-84a.a.-T7 RNAP by sequencing the PT7CAN1 locus. The data demonstrated that the mutation types
generated by PmCDA1-32 a.a.-T7 RNAP and PmCDA1-84 a.a.-T7 RNAP were
basically the same, with C→T mutations accounting for more than 97% and
the remaining 3% being other types of mutations (Fig. 1E), which was
also consistent with the mutation characteristics of
PmCDA1.[16] The distribution of the mutations in
the CAN1 gene was also similar (Fig.1F).Therefore, the length of
linker between PmCDA1 and T7 RNAP has no significant effect on the
mutation effect. Meanwhile, even without UGI, the mutation types
generated by PmCDA1 were really simple, and most of them were C→T
mutations.
DNA-modifying enzymes improved mutation effect
When using PmCDA1-T7 RNAP as the mutator, the strong bias towards C→T
mutations would reduce the diversity of mutants. In cells, the
mismatched U resulting from the deamination of C is excised by
DNA-modifying enzymes to form abasic sites. In the subsequent DNA repair
process, different bases could be randomly inserted into the abasic
sites, resulting in different types of mutations.[34,
35] Thus, we hypothesized that fusing different DNA-modifying enzymes
to PmCDA1 would improve the mutation outcome.
We first chose MAG1 as the DNA-modifying enzyme to link to PmCDA1. MAG1
can remove mismatched bases and initiate base excision repair (BER).[36, 37] Overexpression of MAG1 in cells leads to
an elevated genomic mutation rate.[38] We assumed
that the addition of MAG1 would enhance the excision of mismatched U and
create more abasic sites, thereby generating diverse mutation types
during the subsequent DNA repair process. Since the relative positions
of PmCDA1, T7 RNAP and the DNA-modifying enzymes may influence the
mutation outcome, we designed five expression cassettes with different
constructions and linkers (Fig. 2A). We compared the mutagenic activity
of these fusions with PmCDA1-T7 RNAP and the control strain without
mutagenesis fusions. We found that the construction of the fusion
proteins significantly affects the mutation outcome. The data indicated
that Cons. 3 could raise the mutation frequency up to
1.9x10-3, which was 1.6 to 2 times higher than that of
PmCDA1-T7 RNAP and was the highest among these five fusions. The
mutation frequencies of the other four constructions were about
3x10-4, which was significantly reduced compared with
PmCDA1-T7 RNAP (Fig. 2B). When analyzing the types of mutations, we
found that C→T mutations accounted for 64.5% of the mutations produced
by Cons. 3, followed by G→A mutations (19.2%), C→G mutations (12.5%),
and G→C mutations (3.8%). The proportion of non-C→T mutations is
11-fold higher than that of PmCDA1-T7 RNAP (Fig. 2C). We suspect that
the increase in non-C→T mutations may be due to the enhancement of the
excision of mismatched bases, thus forming more abasic sites-which are
important for BER-and increasing the diversity of mutations. Although
the mutation frequencies of the other four candidates were low, the
mutation types were diverse and most of them were non-C→T mutations. We
speculated that the presence of DNA-modifying enzymes in these
constructions affected the activity of PmCDA1, resulting in the mutation
effect that was apparently different from that of PmCDA1. Considering
the mutation frequency and diversity, we selected the Cons. 3 for
further work.
Based on this, we chose 6 other DNA-modifying enzymes and analyzed their
mutagenic activity (Fig. 3A).[39-44] Among these
candidates, EXO1-PmCDA1-T7 RNAP produced the highest mutation frequency
of 2.2x10-3, which was twice that of PmCDA1-T7 RNAP
(Fig. 3B). When analyzing the mutations generated by EXO1-PmCDA1-T7
RNAP, we found a strong bias towards C→T mutations, similar to PmCDA1.
In 48 randomly selected colonies, C→T mutations accounted for 80.0%,
followed by C→G mutations (11.0%) and other types of mutations (8.0%)
(Fig. 3C). EXO1 is a key enzyme in DNA double-strand break repair,
mismatch repair, and other repair pathways,[43,
45] and we speculated that EXO1 may act synergistically with PmCDA1
to further increase the mutation frequency. In laboratory evolution, a
high mutation rate can greatly accelerate the evolution process. The
mutation frequency generated by REV3-PmCDA1-T7 RNAP was about
1.26x10-3, which was slightly lower than that of
MAG1-PmCDA1-T7 RNAP and EXO1-PmCDA1-T7 RNAP (Fig. 3B), but the mutation
types were diverse, of which C→T mutations accounted for 70.4%,
followed by C→G mutations (14.1%), G→A mutations (9.86%) and other
mutations (5.64%)(Fig. 3C). REV3 involves in DNA translesion synthesis
repair, double-strand break repair, and DNA damage-induced
mutagenesis.[44] Therefore, we hypothesized that,
similar to MAG1, REV3 strengthens the DNA translesion synthesis repair,
in which different bases are inserted into abasic sites, resulting in
multiple types of mutations. In the process of laboratory evolution, the
occurrence of different types of mutations enlarges the mutant spectrum,
and allows us to screen a wider range of desired strains. Different from
cytidine-bearing mutators,[21, 22, 24] after
altering the mutation spectrum by DNA-modifying enzymes, mutations could
occur across all four nucleotides, with G→A or C→G mutations being the
main mutation types, except for C→T mutations, meaning that our system
is able to play a complementary role to the cytidine-based evolutionary
tools.
Dual T7 promoters increased mutation frequency
After changing the constrution of the fusion proteins and adding
DNA-modifying enzymes to improve the mutation effect, the mutagenic
activity of mutators have been improved significantly compared with
PmCDA1-T7 RNAP. Based on this, we inserted two reverse T7 promoters on
both sides of the CAN1 gene and analyzed the mutation effect of
MAG1/EXO1/REV3-PmCDA1-T7 RNAP under this condition (Fig.
4A).[22, 24] We observed that the addition of the
second T7 promoter significantly increased the mutation frequency. In
the dual T7 promoter system, the mutation rate generated by
EXO1-PmCDA1-T7 RNAP could reach 5.13x10-3 after 24 h
of induction, which was 1.57-fold higher than that of the single T7
promoter system (Fig. 4B). With dual T7 promoters, the mutation
frequencies generated by MAG1-PmCDA1-T7 RNAP and REV3-PmCDA1-T7 RNAP
were also significantly increased. After induction for 24 h, the
mutation rates of strains expressing MAG1-PmCDA1-T7 RNAP and
REV3-PmCDA1-T7 RNAP were 3.72x10-3 and
3.26x10-3, respectively, which were
2~2.5-fold higher than that of the single T7 promoter
system (Fig. 4B). We speculated that the dual T7 promoters may increase
the probability of T7 RNAP binding to the T7 promoter, so that
DNA-modifying enzymes-PmCDA1 have a greater chance of acting on the
target gene, leading to higher mutation rates.
When analyzing the mutations produced in the dual T7 promoter system, we
found that the introduction of the reverse T7 promoter had no strong
effect on the mutation types. In the dual T7 promoter system, C→T
mutations generated by MAG1-PmCDA1-T7 RNAP comprised 65.7%, followed by
G→A mutations (13.6%), C→G mutations (11.9%), and other types of
mutations (8.8%). Although the proportion of G→A mutations was slightly
lower than that in the single T7 promoter system, other types of
mutations increased, such as some transversion mutations like G→T. The
mutation types of EXO1-PmCDA1-T7 RNAP in the dual T7 promoter system
were barely changed, among which C→T mutations comprised 82.4%,
followed by C→G mutations (8.33%), G→A mutations (5.1%) and other
mutations (4.17%). In REV3-PmCDA1-T7 RNAP, C→T mutations comprised
73.6%, followed by C→G mutations (12.2%), G→A mutations (12.12%) and
other mutations (2.8%) (Fig. 4C). It can be seen that the dual T7
promoter system had little effect on the mutation characteristics of our
mutagenesis tools, but slightly increased the frequency of some
transversion mutations (such as G→T, G→C, etc.) and made the mutation
types more diverse. Existing deaminase-based evolutionary techniques are
difficult to achieve transversion mutations, and most of them are biased
towards generating specific types of mutations.[22,
24, 25] Therefore, our mutagenesis tools with dual T7 promoters can
further enlarge the mutation libraries, thus promoting the process of
evolution.