bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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A multi-kingdom genetic barcoding system for
precise target clone isolation
Soh Ishiguro1,15,19, Kana Ishida2,19, Rina C. Sakata1,19, Hideto Mori3-5, Mamoru Takana3,
Samuel King1, Omar Bashth1, Minori Ichiraku6, Nanami Masuyama1,4,5, Ren Takimoto1, Yusuke
Kijima1,7, Arman Adel1, Hiromi Toyoshima3, Motoaki Seki3, Ju Hee Oh8, Anne-Sophie
Archambault8, Keiji Nishida9,10, Akihiko Kondo9-11, Satoru Kuhara12, Hiroyuki Aburatani13,
Ramon I. Klein Geltink8, Yasuhiro Takashima6, Nika Shakiba1, and Nozomu Yachie1,3,14,16-18*
1.

School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The
University of British Columbia, Vancouver, Canada.
2. Spiber Inc., Tsuruoka, Japan.
3. Synthetic Biology Division, Research Center for Advanced Science and Technology, The
University of Tokyo, Tokyo, Japan.
4. Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
5. Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa,
Japan.
6. Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
7. Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Tokyo, Japan.
8. BC Children’s Hospital Research Institute, Department of Pathology and Laboratory Medicine,
The University of British Columbia, Vancouver, Canada.
9. Engineering Biology Research Center, Kobe University, Kobe, Japan.
10. Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
11. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe
University, Kobe, Japan.
12. Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu
University, Fukuoka, Japan.
13. Genome Science Division, Research Center for Advanced Science and Technology, The
University of Tokyo, Tokyo, Japan.
14. Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University,
Suita, Osaka, Japan
15. Twitter: @soh__i
16. Twitter: @nzmyachie
17. Twitter: @yachielab
18. Mastodon: sciencemastodon.com/@nzm
19. These authors contributed equally.
*Corresponding author: nozomu.yachie@ubc.ca.
Teaser: A multi-kingdom CRISPR-activatable barcoding system enables the precise isolation of target
barcode-labeled clones from a complex cell population.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Clonal heterogeneity underlies diverse biological processes, including cancer progression, celldifferentiation, and microbial evolution. Cell tagging strategies with DNA barcodes have recently
enabled analysis of clone size dynamics and clone-restricted transcriptomic landscapes ofheterogeneous populations. However, isolating a target clone that displays a specific phenotype
from a complex population remains challenging. Here, we present a new multi-kingdom genetic
barcoding system, CloneSelect, in which a target cell clone can be triggered to express areporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells
are first barcoded and propagated so their subpopulation can be subjected to a given experiment.
A clone that shows a phenotype or genotype of interest at a given time can then be isolated fromthe initial or subsequent cell pools stored throughout the experimental timecourse. This novel
CRISPR-barcode genetics platform provides many new ways of analyzing and manipulating
mammalian, yeast, and bacterial systems.
Cells are not homogeneous in any system. Although they proliferate and replicate the genome that
encodes molecular regulatory programs into their progenies, they also change their statuses according
to the dynamic response of gene expression patterns to environmental signals. As typically shown in
multicellular organisms, cells self-organize through mutual molecular and mechanical communications
and dynamically create complex structures. In these processes, spontaneous mutations in the genome
sometimes impair the cellular program and cause cellular malfunction. Conversely, some othermutations confer growth or survival advantages to the cells, which can be beneficial or catastrophic tothe system.
During cancer chemotherapy, resistant clones arise and expand with unique genetic, epigenetic,
and cellular statuses, contributing to cancer recurrence and metastasis (1-4). In hematopoiesis, stem
cells of an analytically indistinguishable group show fate-restricted differentiation patterns, in whichsome cells seem to be primed for specific lineages by incompletely understood factors (5-8). Similarly,
in vitro stem cell differentiation and direct reprogramming experiments have demonstrated that “elite”
clones reproducibly transform into target cell states (9-11). In laboratory microbial evolution experiments,
different cells within the initial population dynamically expand and shrink their clone sizes through theacquisition of new mutations over multiple generations (12-16).
These views on clonal heterogeneity and cell lineage bias have been rapidly shaped by approaches
that use DNA barcodes and deep sequencing for high-resolution clonal cell tagging. DNA barcoding
involves integrating short and unique variable fragments of DNA—so-called “barcodes”—to individual
cells by lentiviral transduction, transposon-mediated delivery, site-specific DNA recombination in thehost genome, or plasmid transformation. In a given assay, the change in abundance of the barcoded
clones is traced en masse by subsampling the cell populations and quantifying the DNA barcodes by
deep sequencing read counts. Furthermore, the combination of barcode transcription and single-cell
RNA sequencing (scRNA-seq) allows clonal lineages to be analyzed alongside cell states, revealing celllineage-restricted state trajectories (9, 17-23). However, these measurements only provide snapshotsof clone population sizes and gene expression statuses at the time of observation (24), limiting theanalysis of the early causal molecular and/or environmental factors that derive specific fate outcomesof clones.
Did the chemotherapy-resistant clones exist in the initial cell population with the genetic mutations
or altered cell state from the beginning? Did any molecular factor(s) underlie the observed stem celldifferentiation fates? Was the progression of the specific clone conditional on the existence of any otherclones? Flow cytometry cell sorting with immunostaining and emerging image cytometry cell sorting
technologies enable the dissociation of heterogeneous cell populations into single cells with theirobserved phenotypes (25-28) but cannot do the same for a population of clones before they exhibit aphenotype of interest. Furthermore, any current high-content omic measurement requires thedestruction of samples at the time of observation, which precludes the analysis of dynamic molecularand cellular behaviors within the same biological systems. The new concept of “retrospective clone
isolation” has recently emerged to tackle the above-raised questions (29-33). In such a system, abarcoded cell population is first propagated, and its subpopulation is subjected to a given assay (Fig.
1A). After identifying a barcoded clone of interest, the same clone (or its close relative) is isolated in a2

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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barcode-specific manner from the initial or any other subpopulation stored during the experiment. The
isolated live clone can then be subjected to any following experiments, including omics measurements
and reconstitution of a synthetic cell population with the isolates.
The idea of retrospective clone isolation has been implemented using CRISPR–Cas9 genome
editing. In one form of the CRISPR activation (CRISPRa)-based approach (29), cells are tagged with
DNA barcodes that are located upstream of a fluorescent reporter gene with a minimal promoter
sequence. The barcoded cells are designed not to express the reporter gene before the guide RNA(gRNA)-induced transcription activation is triggered. Once a barcoded clone is identified for isolation, agRNA targeting its barcode is introduced to the cell population with catalytically dead Cas9 (dCas9)
fused to transcriptional activator(s) to trigger reporter expression of the target clone. This approach
suffers from low specificity due to leaky expression of the reporter in the non-activated condition,
presumably because of the effect of another constitutively active promoter neighboring the reporter. Thiseffect can be mitigated by barcoding cells with a gRNA library pool and then labeling a target cell by
delivering a plasmid that only encodes a reporter targeted by its corresponding gRNA, with no additional
promoters present (30, 31). However, this strategy prohibits selection of the cells harboring the reporter
plasmid using a secondary selectable marker. The CRISPRa-based approach requires continuous
activation and maintenance of all components when a downstream experiment necessitates reporter
expression in isolated cells. Furthermore, CRISPRa-based retrospective clone isolation has only been
demonstrated in mammalian cell systems.
Genetic circuits based on DNA code alteration generally show highly specific input responses (34).
A wild-type Cas9-based retrospective clone isolation has also been prototyped as a DNA code
alteration-based approach (32). In this system, an out-of-frame start codon is followed by in-frame stopcodons and a DNA barcode is encoded upstream of a reporter gene, whose translation is inactivated.
The reporter translation can be restored by Cas9-induced double-stranded DNA break (DSB) anddeletion through non-homologous end-joining (NHEJ) DNA repair at the barcode region, which
stochastically removes the stop codons and brings the start codon into the coding frame. While thisdoes not usually show unexpected reporter activation for non-target clones and confers permanentlabeling of a target clone, the system’s sensitivity relies on stochastic DNA deletion, which can be abottleneck to efficiency. Cas9-induced DSB is also cytotoxic and potentially impairs the target clone
during the reporter activation procedure (35, 36).
Another approach has been proposed using RNA fluorescence in situ hybridization (FISH), wherebarcoded cells are first fixed, and a target transcribing barcoded clone can be labeled with a fluorophore
probe and isolated by cell sorting (37). While RNA FISH is specific and sensitive, the isolated cells
cannot be used for cell culture-based assays due to the fixation of the analyte.
Here, we report a new CRISPR base editing-based approach, CloneSelect, that overcomes thecurrent technical constraints. CloneSelect is based on the restoration of reporter protein translation bybase editing of an impaired start codon or an upstream stop codon in a barcode-specific manner. The
new method is highly scalable, programmable, and compatible with scRNA-seq. Its specificity markedly
surpasses other CRISPR-based systems. We also present the versatility of the method by implementing
CloneSelect in human embryonic kidney (HEK) cells, mouse embryonic stem cells (mESCs), humanpluripotent stem cells (hPSCs), yeast cells, and bacterial cells.
Results
Mammalian CloneSelect by C→T base editing
CRISPR base editing has widely been used to induce a single nucleotide substitution at a target genomicsite without DSB (38). We first hypothesized that a C→T base editor (CBE)-based circuit (39) would
enable highly sensitive, precise barcode-specific clone isolation with better performance than theprevious CRISPR-based methods. In this CloneSelect C→T system, a barcode is encoded immediately
upstream of a reporter gene whose start codon is mutated to GTG (Fig. 1B left). A constitutively active
promoter transcribes the reporter-encoding region, but the impaired start codon makes its translation
inactive. C→T base editing at the target barcode site enables the antisense strand of the first guanine(cytosine) to be substituted into thymine, restores ATG on the sense strand, and activates the reporter3

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Fig. 1. The CloneSelect circuit. (A) Conceptual diagram of retrospective clone isolation. (B) Different barcodespecific gRNA-dependent reporter activation circuits. CloneSelect C→T, low-copy CRISPRa, and high-copy
CRISPRa. (C) Flow cytometry analysis of single-cell EGFP activation levels. (D) Barcode-dependent reporter
activation of six barcoded cell lines by CloneSelect C→T. Scale bar, 50 µm. (E) Comparison of CloneSelect C→T,
low-copy CRISPRa, and high-copy CRISPRa across the same barcode-gRNA pairs (n=3). For each approach,
Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. (F) CloneSelect A→G.
(G) Comparison of CloneSelect A→G, low-copy CRISPRa, and high-copy CRISPRa across the same barcodegRNA pairs (n=3). Welch’s t-test was performed to compare OT and NT activations. (H) ROC curves along varying
EGFP intensity thresholds for target barcoded cells. Left, CloneSelect C→T and low-copy CRISPRa by the sametargeting gRNAs. Right, CloneSelect A→G and low-copy CRISPRa for the same set of targeting gRNAs. (I)4bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Performance comparison of CloneSelect C→T and CloneSelect A→G. Activated cell frequencies of OT and NT
barcodes were normalized by activated cell frequencies of OT barcodes conferred by low-copy CRISPRa using
the same targeting gRNA. The Mann-Whitney U test was performed to compare the two groups of datasets. *P <0.05; **P < 0.01; ***P < 0.001.

translation. To achieve this, we employed Target-AID, one of the CBEs with a narrow C→T base editing
window in the gRNA target site (36, 38).
To examine the performance of CloneSelect C→T employing EGFP (enhanced green fluorescentprotein) as a reporter, we compared it with two CRISPRa-based approaches. In the low-copy CRISPRa
approach, cells are lentivirally labeled by barcoded CRISPRa reporters, each consisting of a barcode
followed by a minimal promoter and EGFP with an MCP-p65-HSF1 (MpH) transcriptional activator
expression unit encoded on the same vector (Fig. 1B middle). The reporter can be activated byintroducing catalytically dead Cas9 (dCas9) fused to VP64 and a targeting gRNA fused to MS2 hairpins,interacting with the MCP (MS2 coat protein) domain of MpH to recruit transcriptional activators (40). In
the high-copy CRISPRa approach, cells are labeled by gRNAs, in which gRNA spacers serve as
barcodes (Fig. 1B right). Upon transfection of the high-copy CRISPRa reporter-encoding plasmids, thereporter expression can be induced in the cell encoding the targeting gRNA.
CloneSelect C→T was more sensitive than low-copy CRISPRa and more specific than high-copy
CRISPRa in HEK293T cells. For each of the three approaches, we prepared two cell lines with the same
set of barcodes (BC-C1 and BC-C2). CloneSelect C→T with on-target gRNAs activated the reporter
expression in 19.17% (BC-C1) and 17.89% (BC-C2) of target cells while minimizing the activation by
non-targeting gRNAs to less than 0.1% (Fig. 1C and Fig. S1A). Low-copy CRISPRa showed a lower
reporter activation rate of 5.24–6.26% (Fig. 1C and Fig. S1B), and high-copy CRISPRa showed a very
high background activation rate of 52.12–62.15% by non-targeting gRNAs (Fig. 1C and Fig. S1C). The
reporter intensity of activated cells for CloneSelect C→T was slightly but significantly higher (1.76-fold)than that of low-copy CRISPRa (Fig. S1D). The sensitivity of CloneSelect C→T using Target-AID
constructed based on a nickase Cas9 (nCas9) was higher than that constructed based on dCas9 (Fig.
S1E). CloneSelect C→T was also demonstrated to activate the reporter expression in HeLa cells in abarcode-specific manner (Fig. S1F).
To systematically examine the orthogonality between barcodes and gRNAs across the three
approaches, we expanded the analysis to six barcodes (Fig. 1D and Fig. S2A and B) and demonstrated
that CloneSelect C→T and low-copy CRISPRa both specifically activated the reporter expression oftarget barcoded cells, where 2.81–20.46% and 10.39–19.53% of target cells were EGFP positive,
respectively, with minimal non-target activation (Fig. 1E). Consistent with the preceding result,CloneSelect C→T exhibited significantly higher overall reporter expression level than low-copy
CRISPRa (Fig. S2C) and the least false positive rate (Fig. S2D). High-copy CRISPRa showed
substantially lower orthogonality (Fig. 1E and Fig. S2B and D), indicating that this approach is notpractical for retrospective clone isolation. For CloneSelect C→T, we also optimized the input DNA
amount without elevating the minimal false positive rate (Fig. S2E and F).
We also tested a wild-type CRISPR-based system in which the reporter expression interrupted by
an upstream stop codon is removed by the barcode-specific DSB, followed by its stochastic deletion
with NHEJ (Fig. S3A). The reporter activation rate of this approach remained at 2.14–2.30% for target
barcoded cells with a relatively high-false positive rate of 0.51–0.56 % for non-target barcoded cells (Fig.
S3B and C). Additionally, as an alternative to the EGFP reporter, we attempted to develop an mCherry
reporter for CloneSelect C→T (Fig. S4A). However, the mutation of the start codon did not inactivate
the fluorescent expression of the wild-type, presumably because the second methionine served as astart codon (Fig. S4B and C). We found that a stringent barcode-specific gRNA-dependent reporter
activation can be established with the M1V (GTG)+M9A mutant without losing the fluorescence intensity
level (Fig. S4D).
Mammalian CloneSelect by A→G base editing
While exhibiting high efficiency and specificity in human cells, CloneSelect C→T is not applicable forclone isolation of bacterial species as they use GTG to initiate protein translation (41). We thus designed
5bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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another system, CloneSelect A→G, using an adenine base editor ABE-7.10 that induces A→G basesubstitution at the gRNA target sequence (42). In CloneSelect A→G, following a constitutively active
promoter and a start codon, a barcode encoding a TAA stop codon prevents downstream reportertranslation (Fig. 1F). The stop codon can be removed in a gRNA-dependent manner by mutating theantisense strand of the thymine (adenine) to guanine, converting the stop codon into CAA (Proline). We
compared the performance of CloneSelect A→G, low-copy CRISPRa, and high-copy CRISPRa (Fig.
1G and Fig. S5A–F) for three arbitrarily designed barcodes in HEK293T cells. Similar to CloneSelect
C→T, CloneSelect A→G activated the reporter expression for 12.27–31.47% of on-target cells, whereas
the non-target activation was maintained below 0.5% with high orthogonality of barcodes and gRNAs.
Low-copy CRISPRa also exhibited barcode-specific reporter activation, but despite a lower reporter
expression level, its overall false positive rate was higher than CloneSelect A→G (Fig. S5H). High-copy
CRISPRa again showed a high background activation level.
To quantitatively compare the performances of CloneSelect C→T, CloneSelect A→G, low-copy
CRISPRa, and high-copy CRISPRa, we analyzed reporter activation frequencies of on-target barcoded
cells when a stringent false positive rate of 0.5% was permitted. CloneSelect C→T and CloneSelectA→G exhibited 10.05–24.88% and 14.12–35.19%, respectively (Fig. 1H). Low-copy CRISPRa
examined with the same barcode sets also used for CloneSelect C→T and CloneSelect A→G exhibited
2.71–22.37% and 1.92–6.60%, respectively (Fig. 1H). In contrast, high-copy CRISPRa did not show
reporter-activated cells at this threshold. The efficacy of the gRNA to recruit the effector Cas9, in general,
has been known to depend highly on its
targeting sequence (43, 44). To compare
CloneSelect C→T and CloneSelect
A→G, their cell activation frequencies fordifferent
targetbarcodeswere
normalized by those of low-copy
CRISPRa for the same barcodes. As aresult, we did not observe a marked
difference in normalized performance
betweenCloneSelectC→TandCloneSelect A→G (Fig. 1I).
Isolation of barcoded human cells
from a heterogeneous population
To examine if CloneSelect C→T canisolate target barcoded cells from acomplex population, we next generated
a barcoded lentiviral library by pooled
ligation of barcoded EGFP fragments(Fig. S6A). The barcoded EGFP
fragments were amplified by PCR using
an upstream forward primer poolencoding semi-random barcodes and acommon reverse primer (Fig. S6A and
B). The barcode sequences were
designed to be WSNS repeats (W=A or
T; S=G or C) to avoid additional startcodons from appearing. The ligation
product was used to transform
Escherichia coli (E. coli) cells, andtransformant colonies on an agar plate
were scraped to purify a lentiviralbarcode library pool. We estimated thatour protocol normally confers a barcode

Fig. 2. Barcode-dependent cell isolation from a HEK293T cell
population. (A) Nucleotide compositions of barcodes in the
mammalian CloneSelect C→T plasmid mini-pool. Five barcodesthat had unexpected lengths were excluded from thisvisualization. The full barcode sequence list can be found in
Table S1. (B) Barcode abundances in the cell population labeledby the mini-lentiviral barcode pool of CloneSelect C→T. (C)gRNA-dependent labeling of target barcoded cells in a
population. (D) Flow cytometry cell sorting of reporter-activatedcells. (E) Barcode enrichment analysis after cell sorting of thereporter-activated cells. Each row represents the barcodeenrichment profile for each target isolation assay.
6bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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complexity of multiple hundreds of thousands (Fig. S6C) and confirmed most of them to have barcodes
by DNA restriction digestion (Fig. S6D).
To perform a proof-of-principle demonstration, we transformed E. coli cells again with theconstructed library pool, isolated barcoded plasmid clones into a 96-well plate, and pooled 93 that wereconfirmed to have single barcodes by Sanger sequencing (Fig. S6E). The plasmid mini-pool was used
to transduce HEK293T cells with a low multiplicity of infection (MOI) of <0.1, ensuring a single barcode
was introduced to each cell. We amplified the barcode region from the plasmid mini-pool and thetransduced cells by PCR and analyzed them by high-throughput sequencing. We identified 115
barcodes (Fig. 2A) and found that the variation in barcode abundance was replicable in two independent
sequencing library preparations (Fig. S6F) and largely inherited from that in the plasmid pool (Fig. S6G
and H), suggesting no substantial barcode-dependent bias in lentiviral packaging and transduction.
We then tested if we could enrich cells with 16 arbitrarily selected barcodes of different abundancesin the cell population (Fig. 2B). For each target barcode, the cell population was co-transfected with thegRNA and Target-AID plasmids. After four days, we observed EGFP-positive cells in each assay (Fig.
2C) and sorted them by flow cytometry cell sorting (Fig. 2D). For each target barcode, its enrichment in
the sorted cell population was analyzed by PCR and high-throughput sequencing (Fig. 2E). In sum, we
succeeded in enriching the target barcoded cells in 15 out of 16 experiments (93.75%) with a relative
barcode abundance of 29.18–75.75% after sorting. For the 15 successful targets, the mutated startcodon was restored to ATG with an efficiency of 91.63–99.85% (Fig. S6I). A fraction of cells with the
expected barcodes did not demonstrate the GTG→ATG mutation, suggesting that the cytidine
deamination by Target-AID on the antisense strand might be sufficient to express the codon-repaired
reporter transcripts.
Isolation of clones identified in a scRNA-seq platform
To extend the utility of CloneSelect to isolate living clones identified according to their high-dimensionaltranscriptome profiles from a cell population, we made CloneSelect C→T compatible with 3′ capture
scRNA-seq platforms and established scCloneSelect. In scCloneSelect, the barcode located upstreamof the reporter with the mutated start codon (hereafter referred to as “uptag”) is paired with anotherbarcode (“dntag”) downstream of the reporter followed by a hard-coded 30-nt poly(A) sequence (Fig.
3A). The dntag is captured as part of the short-read scRNA-seq reads through the common poly(A)tailed 3′ enrichment strategy (45, 46) and used to refer its corresponding uptag for the reporter startcodon restoration. This change in the circuit design did not affect the reporter activation performance ofCloneSelect C→T in HEK293T cells (Fig. 3B and C and Fig. S7A and B) as well as the high orthogonality
between barcodes and gRNAs (Fig. S7C and D). We also confirmed that dntag barcodes weretranscribed (Fig. S7E) and efficiently captured by a scRNA-seq platform (Fig. S7F).
One intriguing application of scCloneSelect would be to study the fate-determining factors of stem
cell differentiation and cell reprogramming. scCloneSelect can be used to retrospectively isolate, fromthe initial population, cell clones whose states have been identified using scRNA-seq after differentiation.
These clones could then be subjected to further analyses. We therefore tested if the scCloneSelectsystem is functional in selectively labeling target barcoded cells in mouse embryonic stem cell (mESC)
and human pluripotent stem cell (hPSC) populations. In one of our approaches, a constitutively active
Target-AID expression cassette was first stably integrated into a cell population by piggyBac
transposon-based genomic integration, followed by lentiviral transduction of the cells with a
scCloneSelect barcode library (Fig. S7G). The reporter activation was then triggered by introducing atargeting gRNA by lentiviral transduction or lipofectamine transfection. In mESCs, both of the gRNA
reagent delivery methods activated the reporter expression in a barcode-dependent manner with nomarked false positive activation, but lentiviral delivery of the gRNA showed an overall higher cell
activation of 4.57–33.76% in comparison to 2.91–15.77% by the lipofectamine-based plasmid delivery
method (Fig. 3D and E and Fig. S7H). The barcode-specific reporter activation was also successful in
hPSCs, where targeting gRNA reagents were delivered by transfection (Fig. S7I). Additionally, we tested
an approach that required a minimal number of steps, where hPSCs were first lentivirally barcoded, andthe reporter was activated by electroporation with the targeting gRNA and Target-AID delivered together(Fig. S7J). This also enabled barcode-dependent reporter activation in hPSCs (Fig. S7K).
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Fig. 3. Isolation of live stem cell clones characterized by scRNA-seq. (A) scCloneSelect. (B and C) Barcodespecific gRNA-dependent reporter activation of the original CloneSelect C→T and scCloneSelect in HEK293T
cells (n=3). Scale bar, 50 µm. (D and E) Barcode-specific gRNA-dependent reporter activation of three barcoded
mESC lines by scCloneSelect. Target-AID was stably integrated prior to the barcoding. gRNAs were delivered bylentiviral transduction. Scale bar, 100 µm. Welch’s t-test was performed to compare on-target (OT) and non-target(NT) activations. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Schematic diagram of a scCloneSelect workflow to
retrospectively isolate a cell clone demonstrating a gene expression profile of interest from a cell population stored
before they demonstrate the target gene expression pattern. (G) mESC cell culture assays and clone isolation
performed in this work. (H) scRNA-seq of mESC populations treated with LIF and 2i and those without LIF or 2i.
(I) Distribution of cells for arbitrarily selected clones in the two-dimensional embedding of high-dimensional gene
expression space by UMAP (uniform manifold approximation and projection). (J) Abundance of barcoded cellclones in the mESC population. The data was generated based on dntags identified by reamplifying the dntag
reads from the original scRNA-seq libraries. (K) gRNA-specific activation of target barcoded clones in the mESC
population. Scale bar, 50 µm. (L) Barcode enrichment analysis after cell sorting of the reporter-activated cells.
Each row represents the barcode enrichment profile for each target isolation assay. The left heatmap was
expanded from the dashed box area of the right heatmap.
8bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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To demonstrate that barcoded cell clones identified with single-cell transcriptomes can be isolated
from a barcoded cell pool sub-populated in parallel with the one used in scRNA-seq, we set up thefollowing experiments using mESCs (Fig. 3F and Fig. S8A). In this experimental pipeline, a ∆EGFP (nostart codon) fragment reporter is first amplified with forward primers encoding semi-random uptags of
WSNS repeats accompanying a mutated start codon and reverse primers encoding random dntags.
They are cloned into a common backbone plasmid en masse (Fig. S8B and C), and the constructedbarcode plasmid pool is used to lentivirally barcode cells. Barcoded cells are then cultured to propagate
cell clones (Step 1) and separated into three groups of sub-pools (Step 2). The first group is stored forlater clone isolation (Step 3). The second group is used to identify the uptag-dntag combinationreference database by PCR amplification and high-throughput sequencing (Step 4). The last group is
subjected to a given assay, during which intermediate subpopulations can be stored at any point (Step5). A cell population obtained during the assay is then analyzed by scRNA-seq to identify high-content
gene expression profiles of single cells (Step 6). For a single cell demonstrating a cell state of interest,
its dntags can be identified (Step 7), and the corresponding uptag can be retrieved from the uptag-dntag
combination reference database (Step 8). Finally, using a gRNA targeting the identified uptag, thereporter expression of the clone containing the dntag is activated and isolated by cell sorting (Step 9).
Because retroviral transduction, in general, is prone to having chimeric products of input vector
sequences integrated into the genome through its high recombination activity (47, 48), the uptag-dntag
database of the analyte population needs to be determined every time after the pooled transduction.
We optimized the PCR protocol for the amplicon sequencing that identifies this database by minimizing
chimeric PCR artifact products (Fig. S8D and E).
Using a plasmid pool with a barcode complexity of ~150,000, we lentivirally transduced mESCs withTarget-AID at an MOI of <0.1. Following the creation of a clone variation bottleneck by sparse samplingand 10 days of expansion, a subpopulation of the barcoded cells was used to construct the uptag-dntag
database, in which 216 unique barcode pairs were identified (Fig. 3G). After preserving anothersubpopulation, the remaining cells were cultured with serum with LIF and 2i (LIF+2i+) or serum without
LIF or 2i (LIF–2i–), to maintain or to lose pluripotency, respectively. Four days after, scRNA-seq was
performed independently for the two conditions. While the RNA capture rates per cell of the two datasets
were similar (Fig. S9A), the gene expression profiles of single cells were clustered into two distinctgroups along with the culture conditions (Fig. 3H and Fig. S9B). Pluripotency marker genes, such asDppa5a, were highly expressed in the pluripotency maintenance condition, whereas differentiation
marker genes, such as Tpm1, were enriched in the non-pluripotent condition. Although the barcoded
clones did not show a significantly biased distribution between the two conditions, we attempted toisolate the top 10 abundant clones in the scRNA-seq datasets (Fig. 3I and Fig. S9C and D) from theinitial barcoded population. The abundances of these clones varied from 0.0133% to 9.21% in the initial
population according to the analysis determined by the uptag-dntag database (Fig. 3J). Except for one
experiment targeting Clone 153, we obtained a sufficient number of EGFP-positive cells after introducing
the targeting gRNA using lentivirus followed by flow cytometry cell sorting (Fig. 3K and Fig. S9E). For
each of the remaining nine clone isolation attempts, eight showed target clone enrichment with relative
target abundances of 64.8–99.4%, whereas one (Clone 028) showed an enrichment degree of 18.9%(Fig. 3L). Furthermore, we isolated single cells from the EGFP-positive cell samples obtained for Clone
006 and Clone 012, expanded them (Fig. S9F), and confirmed the purification of the isolated clones(Fig. S9G). Accordingly, scCloneSelect was demonstrated to greatly enrich a target clone identified byscRNA-seq from a complex population with a success rate of 80% (eight out of 10).
Yeast CloneSelectClonal barcoding approaches have also been used in microorganisms, such as yeast Saccharomyces
cerevisiae (S. cerevisiae) and E. coli, to study their laboratory evolution and the genomic mutations
accompanying clonal expansions of cells (14, 16). However, the analysis methods have been limited totime-course tracing of clone size dynamics. No technology has been developed to explore genetic,
epigenetic, and other molecular factors contributing to laboratory evolution.
We next extended the CloneSelect C→T system for the yeast S. cerevisiae using mCherry as afluorescent reporter (Fig. 4A). Like the mCherry reporter in mammalian cells, we also needed to truncate
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the first nine amino acids of mCherry to establish the reporter system (Fig. S10A). CBEs, including
Target-AID, developed for mammalian species normally accompany uracil glycosylase inhibitor (UGI)
to inhibit the base excision repair pathway, enhancing both the efficacy and purity of C→T substitution
at the target site (38). However, Target-AID has been tested in yeast without UGI and was demonstrated
to confer C→D (non-C) substitution at the target sequence at a high rate (36). We, therefore, constructed

Fig. 4. Yeast CloneSelect. (A) Yeast CloneSelect C→T circuit. (B and C) Barcode-specific gRNA-dependent
reporter activation. Scale bar, 25 µm. Mean mCherry intensity measured by a plate reader was normalized by
OD595 nm (n=3). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activities. (D)GTG→ATG editing frequencies observed by high-throughput sequencing. Welch’s t-test was performed tocompare OT and NT datasets. (E) Yeast colonies formed on a 10-cm agar plate after performing a target clone
labeling in the yeast cell population of Pool-100. (F–J) Analysis of colonies isolated after clone labeling using eachtargeting gRNA. (F) mCherry positive isolates from Pool-100. (G) mCherry negative isolates from Pool-100. (H)mCherry positive isolates from Pool-1580. (I) mCherry negative isolates from Pool-1580. (J) Summary of theanalysis results. *P < 0.05; **P < 0.01; ***P < 0.001.
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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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a yeast Target-AID with UGI and found that it did not largely impair the base editing activity (Fig. S10B–
D) but as expected, greatly enhanced the frequency of resulting thymine at the target sequence (Fig.
S10E). The efficient yeast CloneSelect C→T reporter activation was only possible by the addition of
UGI (Fig. S10F). Similar to mammalian CloneSelect systems, yeast CloneSelect C→T was also
demonstrated to activate barcoded cells in a highly target-specific manner with a sensitivity of 38.80–
44.13% by co-transformation of Target-AID and gRNA plasmids (Fig. 4B–D and Fig. S10G). Unlike
mammalian cells, the labeled clones could be isolated by picking fluorescent colonies formed on a solid
agar plate (Fig. 4E).
To test the sensitivity of the clone isolation strategy, we generated a barcode plasmid pool by apooled ligation of semi-random barcode fragments to a backbone vector (Fig. S11A and B). Following
a quality control of the pooled cloning reaction (Fig. S11C), E. coli cells were then transformed with theplasmid pool, and either 100 or ~1,580 colonies were pooled to prepare barcode plasmid pools ofdefined complexities (hereafter referred to as Pool-100 and Pool-1580, respectively). After establishing
the barcoded yeast cell populations of Pool-100 and Pool-1580 by yeast plasmid transformation, we
arbitrarily targeted 26 and 31 barcodes of a range of abundances (0.83–5.79% for Pool-100 and 0.12–
0.37% for Pool-1580) to be isolated (Fig. S11D). For each barcoded clone isolation, its corresponding
gRNA plasmid and Target-AID plasmid was co-transformed into the barcoded yeast cells, andfluorescent colonies were isolated, if any, together with four non-fluorescent colonies. The colony
isolates were then cultured in liquid selective media in a microwell plate to measure the fluorescenceintensities by a plate reader and barcode sequences were examined by PCR followed by Sanger
sequencing (Fig. 4F–I). For Pool-100, 16 out of the 26 attempts (61.58%) conferred positive colonies,
all of which, except for one of the three positive colonies obtained for BC 030, had the expectedbarcodes with a GTG→ATG conversion rate of 48.92–97.41% (Fig. 4J). For Pool-1580, six out of the
31 attempts (19.35%) conferred positive colonies, and all of them had the expected barcodes with a
GTG→ATG conversion rate of 81.51–97.20%. The abundance of these successful barcodes in the Pool100 and Pool-1580 yeast pools ranged from 0.120% and 5.78%, respectively, demonstrating that yeast
CloneSelect can also isolate rare clones.
Bacterial CloneSelectLastly, we also established a bacterial CloneSelect system for E. coli clone isolation. We first
implemented CloneSelect A→G using an arabinose-inducible promoter for the EGFP reporter and an
IPTG (Isopropyl β-d-1-thiogalactopyranoside)-inducible promoter for both the gRNA and ABE (Fig. 5A).
The inducible promoter circuits showed an overall higher reporter expression in a barcode-specificmanner (Fig. 5B). At the same time, the EGFP expression level by a targeting gRNA was higher thanthat by a non-targeting gRNA regardless of IPTG induction (Fig. 5C). The expected A→G substitution
was also observed in a target barcode-dependent manner even for the no IPTG condition (Fig. 5D).
These results collectively suggest that the background expression levels of the genome editing reagentswith no inducer were sufficient to confer the base editing. Furthermore, when the EGFP positive control
reporter was used, the arabinose-induced reporter intensity was suppressed by the targeting gRNA (Fig.
5E and Fig. S12A and B), suggesting a transcriptional silencing effect by ABE recruited at the target
site.
Because the fold change in EGFP reporter intensity for the targeting gRNA compared to the nontargeting gRNA was not high (0.901–2.52 fold), we then sought to establish a drug-selectable systemfor bacterial CloneSelect. We first realized that when a Zeocin resistance gene (Sh ble) (49) was used
for the barcode-specific reporter system (Fig. 5F), a constitutively active J23119 promoter conferred cell
growth under the selective condition regardless of the upstream TAA stop codon (Fig. S12C). In contrast,
the arabinose-inducible promoter was selective for the stop codon removal even without the arabinose
supplement, suggesting that a small gene expression level is sufficient for drug resistance. We also
found that targeting gRNA expression from the T7 promoter substantially dropped the number of colonyforming units (Fig. S12D), presumably because nickase Cas9 has known to be toxic to the bacterial
cells (50, 51). Finally, we found that using the same setup as EGFP for the Zeocin resistance reporter
with no inducer condition demonstrated cell growth in a gRNA-dependent manner (Fig. S12E). A

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Blasticidin S-resistance gene (Bsr) (52) also showed utility for gRNA-dependent cell growth with a similar
experimental setup (Fig. S12F and G).
The Zeocin reporter system enabled the isolation of different barcoded strains with high specificity(Fig. 5G). To demonstrate barcoded cell isolation from a complex population, we constructed a pooled
plasmid library for semi-random barcodes of VNN repeats (V=non-T), preventing the appearance of stopcodons (Fig. S13A), and prepared sub-pool libraries by pooling 100 and ~1,550 colonies, respectively(Fig. S13B; hereafter referred as to Pool-100 and Pool-1550). After establishing barcoded E. coli cellFig. 5. Bacterial CloneSelect. (A) Bacterial CloneSelect A→G circuit. ABE and gRNA expressions were
controlled by IPTG-inducible promoters, and the EGFP reporter expression was controlled by an arabinoseinducible promoter. (B and C) EGFP reporter activation of E. coli cells under different inducer conditions. Scalebar, 25 µm. Mean EGFP intensity measured by a plate reader was normalized by OD595 nm (n=3). Welch’s t-test
was performed to compare on-target (OT) and non-target (NT) activities. (D) Base editing outcomes analyzed bySanger sequencing. (E) Activities of the positive control EGFP reporter under the same conditions tested for (C)(n=3). Welch’s t-test was performed to compare OT and NT activities. (F) Zeocin resistance marker-based circuit.
(G) Barcode-specific gRNA-dependent Zeocin resistance reporter activation. (H) Schematic diagram of a bacterial
CloneSelect workflow using a drug selective condition for the target barcoded cell isolation. (I) Abundance ofbarcoded cells in Pool-100 and Pool-1550. (J) Colonies formed on Zeocin-selective and non-selective solid agar
plates after performing the reporter activation of Clone 106 in the E. coli cell population of Pool-100. (K) Analysis
of colonies isolated from Zeocin selective and non-selective plates obtained after clone labeling using eachtargeting gRNA. *P < 0.05; **P < 0.01; ***P < 0.001.
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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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populations of Pool-100 and Pool-1550, we arbitrarily selected target barcodes and attempted to selectcells having each of those barcodes by co-transforming the targeting gRNA and ABE, followed by Zeocinselection (Fig. 5H). We targeted 10 and four barcodes of a range of abundances in Pool-100 (0.047–
2.33%) and Pool-1550 (0.00089–0.211%), respectively (Fig. 5I). In every isolation experiment, theZeocin selective conditions showed a substantially lower number of colonies than the non-selective
conditions (Fig. 5J and K). For each of the successful target barcodes, except for BC 036 of Pool-100
in which we obtained only two colonies in the selective condition, four and four colonies were isolated
from the selective and non-selective conditions, respectively, and their barcodes and base editing
patterns were analyzed by Sanger sequencing. All isolates from the selective conditions had the
expected barcodes and all of the isolates from the non-selective conditions had non-targeted barcodes.
Accordingly, bacterial CloneSelect with the drug resistance marker system demonstrated great
sensitivity and was able to isolate clones that were extremely low in frequency.
Discussion
We describe a method, CloneSelect, which enables the isolation of target barcoded cells from a complex
population using CRISPR base editing. Compared to the CRISPRa and wild-type Cas9-based systems
tested in this study, CloneSelect demonstrated an ability to isolate cells with a higher sensitivity when acertain degree of false positive isolation was permitted. The low-copy CRISPRa experiments confirmed
the reporter expression leak in similar existing methods (29). High background activity observed in thehigh-copy CRISPRa experiments supports the utility of a strategy implemented in the approaches
COLBERT (30) and ClonMapper (31), whereby cells are labeled with gRNAs as barcodes, and target
barcoded clones are activated by provision of a plasmid that encodes only the target gRNAcorresponding reporter with no constitutively active promoters. However, this approach of barcoding
cells using gRNAs does not allow selected cells to maintain reporter activation for subsequent
experiments, when necessary. CloneSelect largely benefits from the precision of base editing and thesimplicity of altering the genetic code, while a CRISPRa-based system involves more additional
endogenous factors to be coordinated for the target barcode-dependent reporter activation. Furthermore,engineering evolutionarily prevalent rules of the genetic code enabled us to implement the same idea
across diverse species. We demonstrated the retrospective isolation of barcoded cells from complex
yeast populations for the first time. While the isolation of barcoded E. coli cells has recently been
demonstrated using barcode-specific CRISPR interference of a counter selection marker (53), we haveshown that bacterial CloneSelect achieves the isolation of target barcoded cells at unprecedented
sensitivity and selectivity.
When practically examining its ability to isolate target barcoded cells from complex populations,
CloneSelect was able to retrieve rare, barcoded cells whose estimated abundances were as small as
0.558%, 0.013%, 0.120%, and 0.00089% in the human HEK293T, mouse ESC, yeast, and E. coli
populations, respectively. The theoretical complexity of semi-random barcodes is limited to 4.19 x 106
and 2.54 x 108 for CloneSelect C→T and CloneSelect A→G, respectively. The sensitivities to obtaintarget barcoded cells that align with the same possible minimal abundance thresholds above are
93.75%,90.00%, 61.56%, and 100.00%, respectively. The limited sensitivity per isolation attempt could
be explained by the general gRNA-dependent genome editing efficacy (43) since isolation success did
not correlate well with the abundance of the target in a population. We suggest that the current sensitivity
range of CloneSelect is sufficient in most prospective assays, with the expectation that multiple
independent clones show a phenotype of interest that allows the user to have multiple attempts. Moreenhanced genetic circuits can be considered to improve sensitivity and specificity. We tested an ORgate with C→T base editing of tandemly arrayed barcodes (Fig. S14A) and an AND-gate with A→Gbase editing of tandemly arrayed barcodes (Fig. S14B). The OR-gate did not function well and enabled
an efficient reporter activation for only one of the three barcodes. The AND-gate enabled the threeinput-dependent reporter activation, but exhibited a tight trade-off with the sensitivity, as expected. Given
the high specificity of the system, introducing multiple independent CloneSelect barcodes per cell might
be a solution to increase the sensitivity if necessary.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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With CloneSelect, new wide-ranging experiments can be conceived in broad fields of biology. A
CloneSelect-barcoded cell population can be subjected to any assay. Existing time-course scRNA-seq
measurement strategies enable interrogation of different clonal lineages in a barcoded population
alongside the dynamic changes in their gene expression landscapes, if the clone population sizes are
not too small (9). In contrast, CloneSelect would allow the clones isolated from different time points fromthe progressing population to be analyzed by diverse approaches (Fig. 6A). Such non-transcriptomic
analyses could include morphological analyses under a microscope and molecular analyses available
for small amounts of input cells, but any currently available methods should be applicable for thedownstream analysis if the isolated clones can be propagated for the given hypothesis.

Fig. 6. New biology directions made possible by CloneSelect. (A) Clonal analysis of molecular profiles in acomplex population. (B) Replication of cell state trajectories. (C) Reconstitution of synthetic cell populations. (D)Transplantation of a fate-mapped clone. (E) New DNA assembly strategy.

Cells isolated by CloneSelect are alive. The clones isolated from the initial stage of a onceperformed assay can be tested to see if they take the same developmental trajectories (Fig. 6B) or used
to reconstitute a synthetic population with another cell population or other isolated clones (Fig. 6C). For
example, a variety of hPSC lines have been reported to be favorable for different cell differentiation andorganoid models (54-58), suggesting that there could also be fate priming of stem cell clones due toundiscovered intrinsic factors. CloneSelect enables mapping of cell states after differentiation and theirsubsequent isolation from the initial population. The fate-mapped stem cell clones could be used toengineer new stem cell-based models or high-quality stem cell therapeutics. Cell clones isolated fromdiverse systems can also be transplanted into animal models (Fig. 6D). Examples include
xenotransplantation of a cancer stem cell clone and aggregation of a fate-mapped stem cell clone withan early embryo. As opposed to the CRISPRa-based system, the barcode-dependent reporter activation
is irreversible. When a fluorescent reporter gene is used, the spatial distribution of the targeted clone
and their interaction with others in a complex biological system can be traced.
In alignment with having DNA sequencing as a readout, CloneSelect would also promote thegenetic engineering of cells and DNA assembly (59-63). Any cell engineering cannot be perfect—only
a fraction of cells that go through an engineering process will harbor the target genetic product.
Therefore, obtaining successful cells becomes difficult when their fractions in the reaction pool are verysmall. In such a task, through the barcoding and sequencing of the product cells, CloneSelect could
retrieve target cells with the desired genetic engineering outcome. We also envision CloneSelect tolargely improve DNA assembly in general (Fig. 6E). Currently, it is common practice to transform a DNA
assembly reaction sample into E. coli cells in order to clone each assembly product, followed by colony
isolation and sequencing-based confirmation. A pool of CloneSelect barcodes can be used tomolecularly tag DNA assembly products. In this framework, the assembly products are used to transformE. coli, followed by the pooling of transformants and extraction of the pooled plasmid products from its
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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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subpopulation. The pooled plasmid product is then sequenced by long-read DNA sequencing to identify
barcodes assigned to the desired product. Finally, the clone harboring the target product can be isolatedusing CloneSelect in a target barcode-specific manner. This strategy would enable the utilization of eveninefficient DNA assembly protocols.
Accordingly, CloneSelect is a new way to precisely isolate target cell clones from complex
populations. Its performance demonstrated in multi-kingdom species opens a vast array of possibilities
to answer complex questions in diverse areas of biology.
Acknowledgments
We thank the members of the Yachie lab at the University of British Columbia and the University ofTokyo, especially Sanchit Chopra, Sean Okawa, Sofia Romero, Yin Liu, Yeganeh Dorri, Madina Kagieva,
and Rina Yogo, for their constructive feedback. We also thank Kaori Shiina, Shiro Fukuda, and Tara
Stach for their technical support in high-throughput sequencing and Andy Johnson for the flow cytometrycell sorting. This study was funded by the New Energy and Industrial Technology Development
Organization (NEDO), the Japan Agency for Medical Research and Development (AMED), the Japan
Science and Technology Agency (JST), the Takeda Science Foundation, the SECOM Science andResearch Foundation, the Canada Foundation for Innovation (CFI), the Canadian Institutes of HealthResearch (CIHR) CoVaRR-Net Project (all to N.Y.), and the Allen Institute Allen Distinguished
Investigator Award (to N.Y. and N.S.). Authors were also supported by the JST PRESTO program (N.Y.),
the Canada Research Chair program (N.Y.), the Ryoichi Sasakawa Young Leaders Fellowship (S.I.),the Taikichiro Mori Memorial Research Fund (S.I. H.M., and N.M.), the Japan Society for the Promotionof Science (JSPS) DC Fellowship (S.I., H.M. Y.K., and N.M.), the JSPS Overseas Research Fellowships(S.I.), Japan Student Services Organisation (R.S.), the Funai Overseas Scholarship (R.S.), theTakenaka Scholarship Foundation (R.T.), the Ministry of Science and Education (MEXT) Research
Scholarship (A.A.), the Natural Sciences and Engineering Research Council of Canada (NSERC)
Undergraduate Student Research Award (S.King), Michael Smith Health Research BC Scholar Award(R.I.K.G.), and the Michael Smith Health Research BC Trainee Award (A.-S.A). High-throughput
sequencing data analysis was performed using the SHIROKANE Supercomputer at the University ofTokyo Human Genome Center.
Author contributions
S.I. and N.Y. conceived the study. S.I., R.S., and M.T. performed all the major mammalian cell
experiments. K.I. and M.T. performed all the yeast and bacterial experiments. O.B., N.S., M.I., and Y.T.
performed a part of the experiments using mESCs and hPSCs. S.I., H.M., H.T., R.S., Y.K., and H.A.
performed the high-throughput sequencing analyses. R.T., A.A., and N.M. performed preliminary assays
and contributed to the design of the system. J.H.O., A.-S.A., and R.I.K.G. contributed to the optimizationof the system. S.King supported the plasmid construction. K.N., A.K., and S.Kuhara also provided
substantial ideas in designing the proposed system. M.S. supported the flow cytometry cell sorting. H.A.
supported the high-throughput sequencing, the flow cytometry analysis, and the microscope imaging.
S.I., R.S., K.I., and N.Y. wrote the manuscript.
Competing interests
K.I. is an employee of Spiber Inc. The other authors declare no competing interests.

15

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Materials and Methods

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Oligonucleotides were chemically synthesized by FASMAC, Integrated DNA Technologies, or Eurofins
Genomics. All the oligonucleotides and cloning procedures used to construct the plasmids in this study
can be found in Table S2. We used QUEEN version 1.2.0 (https://github.com/yachielab/QUEEN) to
describe each plasmid construction and generate the annotated plasmid files in the GenBank (gbk) fileformat, embedding the full construction procedure (Table S2). A QUEEN-generated gbk file behaves asa quine code, where the QUEEN simulation code that generated the gbk file can perfectly bereconstructed from the file itself (64). We believe that providing the QUEEN-generated gbk files satisfies
the mandate of reporting the reproducible protocols for the newly constructed plasmids. We also
provided natural language descriptions for each plasmid construction step in the QUEEN code, allowing
the user to retrieve the natural language description of materials and methods for each plasmid bysimply executing "QUEEN --protocol_description --input [gbk file]” in a QUEEN-installed environment. A
custom QUEEN wrapper that generated all QUEEN-generated gbk files can be found athttps://github.com/yachielab/CloneSelect_v1/tree/main/QUEEN. We, therefore, do not provide theplasmid construction protocols here. All plasmid DNA sequences were confirmed by Sanger sequencing.
The representative plasmids are in the process of being deposited to Addgene with their QUEENgenerated gbk files, which Addgene agreed to accommodate.

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Cell culture
HEK293T and HeLa cells. Human embryonic kidney 293Ta (HEK293Ta) and HEK293T Lenti-X cells
were purchased from GeneCopoeia (#LT008) and Takara (#632180), respectively. Cells were cultured
in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich #11965084) supplemented with 10%
Fetal bovine serum (FBS) (Gibco #16000044) and 1% Penicillin-Streptomycin (Wako #168-23191) at
37ºC with 5% CO2 in a cell culture incubator. Cells were detached and passaged using 0.25 w/v%
Trypsin-EDTA (Wako #203-20251) once the cells reached 70–90% confluency. The cell lines wereregularly tested for mycoplasma contamination. When microscope imaging of HEK293T cells was
performed with Hoechst 33342 (Invitrogen #H3570) counterstain, 100–200 µL of Collagen-I (Nippi#PSC-1-100-100) diluted in 5 mM acetic acid was added to each cell culture plate well, incubating for
30 min at 37ºC. The collagen-coated plate wells were washed by 100–200 µL of 1x PBS before use.

Plasmids

Mammalian CloneSelectmESCs. Under the approval by the Institutional Animal Care Committee of the University of Tokyo(RAC180003), mESCs were established from embryos of a 129(+Ter)/SvJcl (female) x C57BL/6NJcl(male) cross and maintained in Dulbecco’s Modified Eagle Medium - low glucose (Sigma-Aldrich#D6046-500ML) supplemented with 1% Penicillin-Streptomycin (Gibco #15140122), 1% MEM Nonessential Amino Acids (Wako #139-15651), 1% GlutaMAX Supplement (Gibco #35050061), 1% Sodium
Pyruvate (Gibco #11360070), 15% FBS (Gibco #16000044), 100 µM/mL 2-Mercaptoethanol (Wako#131-14572), 103 units/mL ESGRO Recombinant Mouse LIF Protein (Millipore #ESG1107), 3.0 µM
CHIR99021 (GSK-3 inhibitor) (Wako #038-23101), and 1.0 µM PD0325901 (MEK inhibitor) (Toris#4423). A sufficient volume of 0.1% Gelatin (Sigma-Aldrich #G9391) in 1x Phosphate-buffered saline(PBS) (Takara #T9181 or Gibco #70011044) was added to each well such that the liquid could cover
the entire surface and be aspirated after one hour at 37ºC before plating cells. Cells were cultured in acell culture incubator at 37ºC with 5% CO2. The cell culture medium was replaced at least once every
two days. The cell line was regularly tested for mycoplasma contamination.
hPSCs. CA1 human PSC (hPSC) line was used after approval by the Canadian Institutes of HealthResearch Stem Cell Oversight Committee. CA1 hPSCs were cultured using mTeSR Plus (STEMCELLTechnologies #100-0276) cell culture medium in a humidified incubator at 37ºC with 21% O2 and 5%
CO2. Culture plates were coated with Geltrex LDEV-Free Reduced Growth Factor Basement Membrane16bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Matrix (Gibco #A1413201). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)(Gibco #11320033) was used to make a working solution of Geltrex (Gibco #A1413201) with 1:100
dilution. A sufficient volume of Geltrex was added to each well such that the liquid could cover the entire
surface and be aspirated after one hour incubation at 37ºC before plating cells. The cell culture medium
was exchanged every other day after plating the cells. Regularly, cells were passaged as medium-sizedclumps. Upon aspiration of the media, ReLeSR (STEMCELL Technologies #05872) was added, andcells were incubated at room temperature for about 1 min before the second aspiration. Cells were thenplaced inside the incubator for 4-5 min, added fresh media, and dissociated by pipetting up and down.
Cells were then plated and placed inside the incubator. For single-cell passaging, we used TrypLE
Express (Gibco #12604021). Cells were then placed inside the incubator for four min before adding asufficient fresh media to stop the activity of TrypLE Express. Cells were then collected in centrifuge
tubes, dissociated by pipetting up and down, and passed through a 40 µm cell strainer (Sarstedt#83.3945.040) to remove cell clumps. The tubes were then centrifuged at 300–400 g for 5 min and thesupernatant was aspirated. The pellets were resuspended in a fresh media supplemented with 10 µM
ROCK inhibitor Y-27632 (Tocris Bioscience #1254) for 24 hours to promote the survival of the single
cells. For culturing H1 hPSCs, we used StemFit AK02N cell culture medium (REPROCELLAHS#RCAK02N) with Y-27632 (Cayman #10005583) added for one or two days after plating. Culture plates
were coated with the recombinant Laminin-511 E8 fragment using iMatrix-511 Silk (MAX #892021). The
cell lines were tested for mycoplasma contamination.
Mammalian CloneSelect barcode libraries
CloneSelect C→T barcode library. To generate the CloneSelect C→T barcode library, a semi-randomoligo pool SI#679 encoding 5′-CCGWSNSWSNSWSNSWSNSNGTG-3′ was first chemicallysynthesized (Table S2), where the antisense strand sequence of the 5′-CGG-3′ PAM sequence and aquadruple repeat of WSNS (W=A or T; S=G or C) were followed by a mutated start codon (GTG). The
WSNS repeat restricts additional start codons from appearing before the downstream reporter. An
EGFP coding sequence was then amplified from pLV-eGFP (Addgene #36083) in 25 separate PCR
reactions, each in 50 µL volume, composed of 1 ng/µL of pLV-eGFP template plasmid, 1.25 µL of 20µM SI#679 oligo pool as a forward primer,1.25 µL of 20 µM SI#680 as a common reverse primer, 0.5µL of Phusion High-fidelity DNA Polymerase (NEB #M0530), 10 µL of 5x Phusion HF Buffer (NEB
#B0518S), and 5 µL of 2.5 mM dNTPs (Takara #4025), with the following thermal cycle condition: 98ºC
for 30 s, 30 cycles of 98ºC for 10 s, 72ºC for 10 s, and 72ºC for 60 s, and then 72ºC for 5 min for the
final extension. The amplified fragment was digested by DpnI (NEB #R0176) for an hour at 37ºC, pooled
into a single tube (1,250 µL in total), and column purified using FastGene PCR/Gel Extraction Kit (Nippon
Genetics #FG-91302). The purified fragment was then subjected to EcoRI-HF (NEB #R3101S) and XbaI(NEB #R0145S) digestion overnight at 37ºC and purified again by FastGene PCR/Gel Extraction Kit(Nippon Genetics #FG-91302). To obtain a highly complex lentiviral plasmid pool, we performed a totalof five ligation reactions using PCR strip tubes, each for a 50-µL reaction containing ~30 fmol of EcoRIXbaI digested pLVSIN-CMV-Pur backbone plasmid (Takara #6183), ~300 fmol of the insert fragment,
2.5 µL of T4 DNA Ligase (NEB #M0202), and 5 µL of 10x T4 DNA Ligase Buffer (NEB #B0202). The
reaction samples were incubated at room temperature for two hours and purified by FastGene PCR/Gel
Extraction Kit (Nippon Genetics #FG-91302). The ligation sample was then used to transform NEB
Stable Competent E. coli cells (NEB #C3040I). The transformation was performed in five reactions, eachwith 1,250 ng of the ligation sample transformed to 200 µL of the competent cells according to the
manufacturer’s high-efficiency transformation protocol. Following one-hour outgrowth in SOC medium(NEB #B9020) at 37ºC, cells were spun down and plated on a total of 25 LB agar plates containing 100µg/mL Ampicillin (Wako #014-23302). After overnight incubation at 37ºC, colonies formed on the plates
were scraped by adding 1–2 mL ddH2O, pooled into a flask, and further incubated with 200–300 mL of
LB liquid medium containing 100 µg/mL Ampicillin (Wako #014-23302) overnight at 37ºC. We plated thetransformation sample on agar plates with 500-fold dilution in triplicate and estimated the barcode
complexity of the constructed library to be ~6.8 x 105. The plasmid library was finally purified by
NucleoBond Midi-prep Kit (Macherey-Nagel #740410) and stored at –20ºC before use. We performed
the isolation of 16 random clones and triple restriction enzyme digestion using BsrGI-HF (NEB
17

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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#R3575S), ClaI (NEB #R0197S), and PvuI-HF (NEB #R3150S), and confirmed that all the tested clones(16/16) contained the expected barcode inserts. To generate a low-complexity library for the proof-ofconcept assays using HEK293T cells, we examined barcode sequences of 96 isolated clones by Sanger
sequencing using a sequencing primer SI#471. After removing three clones observed to have mixed
Sanger sequencing spectra in the barcode region, barcoded plasmids were pooled with an equimolar
ratio and subjected to high-throughput sequencing and lentiviral packaging.
scCloneSelect barcode library. The scCloneSelect barcode library was prepared similarly to the
CloneSelect C→T barcode library. An EGFP coding sequence was first amplified by PCR from pLV-CS112 (Addgene #131127) using the semi-random oligo pool SI#679 as a forward primer and another oligopool RS#244 as a reverse primer. The PCR was performed in 25 separate reactions, each in 40 µL
reaction volume, composed of 0.12 µL of 10 ng/µL pLV-CS-112 template plasmid, 2 µL each of forward
and reverse primers, 0.6 µL of Phusion High-fidelity DNA Polymerase (NEB M0530), 8 µL of 5x Phusion
HF Buffer (NEB #B0518S), and 3.2 µL of 2.5 mM dNTPs (NEB #N0447) with the following thermal cycle
condition: 98ºC for 30 s, 30 cycles of 98ºC for 10 s, 65ºC for 10 s, and 72ºC for 60 s, and then 72ºC for
5 min for the final extension. The amplified barcode-EGFP fragment was pooled into a single tube (1,000µL in total) and digested with 12.5 µL of DpnI (NEB #R0176) at 37ºC for 1 hour. The digested productwas then size-selected using FastGene PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). The
purified product was subjected to EcoRI-HF (NEB #R3101S) and XbaI (NEB #R0145) digestion
overnight at 37ºC and purified again by FastGene PCR/Gel Extraction Kit (Nippon Genetics #FG-91302).
For backbone preparation, 25 µg of pRS193 lentiviral cloning backbone plasmid was digested by EcoRIHF (NEB #R3101S) and XbaI (NEB #R0145) at 37ºC overnight and size-selected using FastGene
PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). 1.25 µg of the digested backbone, 320 ng of thepurified insert, 25 µL of T4 DNA Ligase (Nippon Gene #317-00406), and 25 µL of 10x T4 DNA Ligase
Buffer (NEB #B0202) were mixed in a total volume of 250 µL and incubated overnight at 16ºC. The
ligation sample was then used to transform NEB Stable Competent E. coli cells (NEB # C3040I). The
transformation was performed in a total of 17 reactions, each with 4 µL of the ligation sample
transformed to 50 µL of the competent cells according to the manufacturer’s high-efficiency
transformation protocol. Following one-hour outgrowth in SOC medium (NEB #B9020) at 37ºC, cells
were spun down and plated on a total of 15 LB agar plates containing 100 µg/mL Ampicillin (Wako #01423302). After overnight incubation at 37ºC, colonies formed on the solid agar plates were scraped byadding 1–2 mL ddH2O, pooled into a flask, and further incubated with 200–300 mL of LB liquid medium
containing 100 µg/mL Ampicillin (Wako #014-23302) overnight at 37ºC. We plated the transformation
sample on agar plates with 300-fold dilution in duplicate and estimated the barcode complexity of theconstructed library to be ~1.5 x 105. The plasmid library was finally purified by NucleoBond Midi-prep
Kit (Macherey-Nagel #740410) and stored at –20ºC before use. We performed the isolation of 20
random clones and confirmed the expected fragment insertion for 17/20 by genotyping PCR using theprimer pair RS#147 and SI#514. From the same 20 clones, we selected six (including the three that did
not yield the expected genotyping PCR bands), performed double digestion using EcoRI-HF (NEB
#R3101S) and BamHI-HF (NEB #R3136S), and Sanger sequencing using sequencing primers SI#514
and RS#147 for uptag and dntag, respectively, and confirmed that all the tested clones contained the
expected uptag and dntag inserts.
Lentiviral barcoding
Virus packaging. HEK293T cells were plated on either a 10-cm cell culture dish at a density of ~2 x 106
cells with 10 mL of the culture medium or 6-well cell culture plate wells at a density of ~2 x 105 cells/wellwith 2 mL of the culture medium one day before plasmid transfection. For packaging using a 10-cm cell
culture dish, 3.0 µg transgene vector, 2.25 µg psPAX2 (Addgene #12260), 0.75 µg pMD2.G (Addgene#12259), and 18 µL of 1 mg/mL PEI MAX (Polysciences #24765-100) were dissolved in 1,000 µL of 1x
PBS and applied to the cell culture. For packaging using a 6-well cell culture plate well, 489 ng transgene
plasmid, 366.7 ng psPAX2, 122.3 ng pMD2.G, and 2.93 µL of 1 mg/mL PEI MAX (Polysciences #24765100) were dissolved in 300 µL of 1x PBS and applied to the cell culture. The culture medium waschanged to a fresh medium one day after transfection. The transfected cells were further incubated for18bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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48–72 hours. The cell culture supernatant was then harvested and filtered with 0.22 µm pore size sterile
syringe filters. The recombinant lentivirus sample was then aliquoted 500–1,000 µL each into 1.5-mL
test tubes and stored at –80ºC.
Virus concentration. To increase virus infection titer, we concentrated harvested virus samples using apolyethylene glycol (PEG)-based method (65) using PEG 6000 (Wako #169-09125) or Lenti-X
Concentrator (Takara #631231). When PEG 6000 was used, ~10 mL of recombinant virus sample was
mixed with 2.55 mL of 50 w/v% PEG 6000, 1.085 mL of 4M NaCl, and 1.365 mL of 1x PBS in a 50 mL
tube. The sample was continuously mixed using a rotator at 4ºC for 90 min and centrifuged at 4,000 g
and 4ºC for 20 min. The supernatant was discarded, and the precipitated virus pellet was resuspended
with 1.1 mL of Opti-MEM (Gibco #31985062) by pipetting and vortexing until fully dissolved, resulting in
a 10-fold concentration of the virus sample. The virus concentration using Lenti-X Concentrator was
performed with the manufacturer’s protocol and dissolved in Opti-MEM (Gibco #31985062) to yield a10- or 15-fold concentration. The concentrated virus samples were stored at –80ºC.
Transduction of HEK293T and HeLa cells. Cells were seeded on 6-well cell culture plate wells at adensity of ~2 x 105 cells/well with 2 mL of the culture medium one day before transduction. A total of
1,000 µL transduction mix containing 1 µL of 2 µg/mL Polybrene (Sigma-Aldrich #TR-1003),
recombinant lentivirus, and the cell culture medium was applied to each well alongside non-virus
controls. One day after transduction, cells were trypsinized and plated on 96-well cell culture plate wells
at a density of ~5 x 103 cells/well for a virus titer measurement. The next day, the culture medium was
exchanged with a fresh medium containing 2.0 µg/mL Puromycin (Gibco #A1113803) or 5.0 µg/mL
Blasticidin S (Wako #029-18701) to select the infected cells for two to five days. After drug selection,
cell viability was measured using CellTiter-Glo (Promega #G7570) according to the manufacturer’s
protocol. The luminescence was quantified using Infinite 200 PRO plate reader (TECAN). The
background luminescence from wells without cell samples was used to subtract the signals. For eachcondition, the multiplicity of infection (MOI) was determined by the fraction of the survived cells
compared to the non-selective condition control. The samples with an MOI close to but not over 0.1 wasused for the following analyses, where most of the selected cells were expected to have a single viral
integration according to Poisson statistics.
Transduction of mESCs. Cells were seeded on 6-well cell culture plate wells at a density of ~2 x 105
cells/well with 2 mL of the culture medium one day before transduction. For cell transduction,
recombinant virus samples with volume of 10–100 µL were thawed on ice, mixed with 1.5 µL of 8 µg/mL
Polybrene (Sigma-Aldrich #TR-1003) and 1.5 mL of fresh cell culture medium, and applied to the cells.
To select the transduced cells, the culture media was exchanged with a fresh media containing 1.0µg/mL Puromycin (Gibco #A1113803) two days after infection, followed by incubation for three days.
Survived cells were detached, and cell counts were measured using Automated Cell Counter TC20
(BioRad). The MOI was determined by the fraction of the survived cells compared to the non-selective
condition control. The samples with an MOI close to but not over 0.1 was used for the following analyses.
Transduction of hPSCs. Cells were seeded on 6-well cell culture plate wells at a density of ~1 x 105
cells/well with 2 mL of the culture medium one day before transduction. For cell transduction,
recombinant virus samples with volume of 10–100 µL were thawed on ice, mixed with 1.5 µL of 8 µg/mL
Polybrene (Sigma-Aldrich #TR-1003) and 1.5 mL of fresh cell culture medium, and applied to the cells.
After 48 hours of infection, the culture media was exchanged with a fresh media containing 1.0 µg/mLof Puromycin (Gibco #A1113803) for three days. The reporter-integrated cells were dissociated into
single cells and subjected to flow cytometry cell sorting to enrich EGFP-negative cells. The sorted cells
were maintained with StemFit AK02N culture media (REPROCELL #RCAK02N).
Preparing cells with stably integrated Target-AID
mESCs. The mESC line with stably integrated Target-AID was established by electroporation using
NEPA21 Super Electroporator (NEPAGENE). After detaching cells from cell culture plate wells, ~2 x 106
19bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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cells were mixed with 100 µL of Opti-MEM (Gibco #31985062), 2.0 µg of pNM1325, and 0.7 µg of aSuper piggyBac transposase vector (SBI #PB210PA-1), and transferred to an electroporation cuvette(NEPAGENE #EC-002S). The electroporation was done by two poring pulses of positive electric polarity
with 115 V for 5 ms with 50-ms intervals and 10% decay rate, and five transfer pulses each for positive
and negative electric polarities with 20 V for 50 ms with 50-ms intervals and 40% the decay rate. Afterelectroporation, cells were transferred to a 10-cm cell culture dish with a fresh culture medium. The
medium was exchanged with a fresh medium one day after electroporation. Two days post
electroporation, the medium was exchanged again with medium containing 5 µg/mL of Blasticidin S(Wako #029-18701) to select cells with stable integration. The cells were incubated for about two weeks
in the selection medium.
hPSCs. To establish an hPSC line with stably integrated Target-AID, CA1 cells were seeded on 24-well
cell culture plate wells at a density of ~5 x 104 cells/well with 1 mL of the culture medium one day before
transfection. The next day, the medium was exchanged to remove Y-27632. The transfection mix was
prepared by combining 450 ng of pNM1325 (CAGp-Target-AID-2A-Blast), 50 ng of a hyperactive
PiggyBac transposase plasmid, 1 µL of Lipofectamine Stem Transfection Reagent (Invitrogen#STEM00001), and 49 µL of Opti-MEM (Gibco #31985062) and applied to the wells after 10 min
incubation. The next day, the culture medium was exchanged with a fresh medium to remove thetransfection reagent. Three days after transfection, the medium was exchanged with a fresh medium
containing 5 µg/mL of Blasticidin S to start selection for 24 hours. Another selection was performed twodays until confluent and cells were passaged into a new cell culture plate. One extra selection was
conducted to ensure a positive selection of the cells.
Transient plasmid delivery
Transfection of HEK293T and HeLa cells. Cells were seeded on 24-well cell culture plate wells at adensity of ~5 x 104 cells/well with 500 µL of the culture medium or 6-well cell culture plate wells at adensity of ~2 x 105 cells/well with 2,000 µL of the culture medium one day before transfection. For regulartransfection in a 24-well plate, a total of 400 ng of plasmids (3:1 volume of Cas9-based enzyme plasmid
to gRNA plasmid when they were mixed), 1.2 µL of 1 mg/mL PEI MAX (Polyscience #24765), and 100µL of 1x PBS were mixed, incubated for 5 or 10 min at room temperature, and applied to each well. The
dose-dependent activation assay with the different Target-AID expression plasmid was performed using
a 24-well plate with the plasmid amount per well ranging from 50–800 ng and 1 mg/mL of PEI MAX
whose volume was adjusted to be 3 µL per 1 µg of the plasmid. For isolating barcoded HEK293T cells
using CloneSelect C→T, we used 6-well cell culture plates, and a total of 800 ng of plasmids encoding
both Target-AID and gRNA were combined with 2.5 µL of 1 mg/mL PEI MAX (Polyscience #24765) and200 µL of 1x PBS and applied to each well after 5–10 min incubation at room temperature.
Transfection of mESCs. Cells were seeded on 48-well cell culture plate wells at a density of ~6 x 104
cells with 200 µL of the culture medium. For each reaction, a total of 200 ng of plasmids were diluted in
20 µL of Opti-MEM (Gibco #31985062), and 0.6 µL of Lipofectamine 2000 (Invitrogen #11668019) was
combined with 19.4 µL of Opti-MEM (Gibco #31985062) as a transfection mix. The plasmid andtransfection mix were then combined and applied to each well after 5 min incubation at room
temperature.
Electroporation of hPSCs. For the gRNA-dependent reporter activation of the barcoded CA1 hPSCs
with the stably integrated Target-AID, we used Neon Transfection System (Invitrogen MPK5000) to
deliver the gRNA plasmid by electroporation. Cells were detached from cell culture plate wells, and ~1
x 105 cells were mixed with 100 µL of Neon Resuspension Buffer and 2.0 µg of gRNA plasmid. The
electroporation was done by 1,200 V for 30 ms with one pulse. In the co-delivery of Target-AID andgRNA expression plasmids to the barcoded H1 hPSCs, ~1 x 105 cells were mixed with 100 µL of Neon
Resuspension Buffer, 3.0 µg of Target-AID plasmid, and 3.0 µg of gRNA plasmid, and theelectroporation was done by 1,200 V for 20 ms with two pulses.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Preparing microscope imaging samples
For imaging HEK293T cells, 25 µL of 0.1 mg/mL Hoechst 33342 (Invitrogen #H3570) dissolved in DMEM
was directly added to each well of 24-well cell culture plates three days after transfection for nuclear
counterstaining. The specimens were incubated at room temperature for 10 min, followed by removalof the culture medium. Cells were gently washed with fresh 500-µL of DMEM once and filled with 500µL of fresh DMEM before imaging. For imaging HeLa cells, cells were first washed with 500 µL of 1x
PBS, added 25 µL of 0.1 mg/mL Hoechst 33342 dissolved in 475 µL of DMEM and incubated at room
temperature for 10 min. Cells were gently washed with 500 µL of 1x PBS and filled with 500 µL of fresh
DMEM. For mESCs, 5.0 µg/mL Hoechst 33342 (Invitrogen #H3570) dissolved in the cell culture medium
was directly added to each well and incubated at room temperature for 10 min to proceed with imaging.
All live cell imaging was performed using BZ-X710 (Keyence), InCellAnalyzer 6000 (GE Healthcare), or
IX83 (Olympus) with a 4x, 10x, or 20x objective lens. The contrast and brightness of the images obtainedin a single batch of the experiment were uniformly adjusted using ImageMagick (Version 7.1.0-20) orFiji (Version 1.0).
Flow cytometry analysis
Cells were detached by 0.25 w/v% Trypsin-EDTA (Wako #201-18841), incubated at 37ºC for 5 min,collected into a 1.5-mL tube or a 96-well round-bottom plate, and centrifuged at 1,000 rpm and room
temperature for 5 min. After aspirating the supernatant, cell pellets were gently resuspended with 150–
500 µL of ice-cold FACS buffer consisting of 2% FBS in 1x PBS. The samples were immediately placedon ice until flow cytometry analysis. The flow cytometry analysis was performed with BD FACSVerse
Cell Analyzer (BD Biosciences) or CytoFLEX Flow Cytometer (Beckman Coulter). The samples weremixed gently by pipetting or vortexing immediately before the analysis. Approximately 10,000–20,000
raw events were acquired for each sample. The data analysis was performed with custom R scripts
using flowWorkspace (version 0.5.40) (https://github.com/RGLab/flowWorkspace), flowCore (version
1.11.20)(https://github.com/RGLab/flowCore)
and
CytoExploreR(version
1.1.00)(https://github.com/DillonHammill/CytoExploreR) or a Python package FlowCytometryTools (version
0.5.0) (https://github.com/eyurtsev/FlowCytometryTools). Codes are available athttps://github.com/yachielab/CloneSelect_v1/tree/main/FACS.
Flow cytometry cell sorting
HEK293T cells. Four days after the transfection of Target-AID and gRNA plasmids for barcode-specificcell isolation, cells were detached by 0.25 w/v% Trypsin-EDTA (Wako #201-18841), incubated at 37ºC
for 5 min, collected into a 1.5-mL tube, and centrifuged at 1,000 rpm and room temperature for 5 min,followed by resuspension into a 5-mL polystyrene round-bottom tube (FALCON) containing 150–500 µL
of 1% FBS in 1x PBS. The cell suspension was immediately placed on ice until sorting. The sorting was
performed using BD FACSJazz (BD Biosciences) with 1.0 Drop Single Sort mode. Cells were first gatedusing FSC-A and SSC-A, and the gate for EGFP+ cells was determined to obtain those having high
FITC-A intensities that were not observed in a control cell sample transfected with Target-AID and NT
gRNA plasmids. EGFP+ cells were sorted into 8-strip PCR tubes (Nippon Genetics #FG-018WF), eachcontaining 2.5 µL of 1x PBS. For good cell recovery, the cell destination position in the collecting tube
was adjusted manually for each sample. The sample was immediately placed on an ice-cold 96-wellaluminum block. Although the EGFP+ cell rate varied across the samples, approximately 50–600
EGFP+ cells were recovered from each experiment.
mESCs. Three days after the transduction of a query gRNA, each cell sample was expanded in a 10cm cell culture dish. Cells were detached by 0.25 w/v% Trypsin-EDTA (Gibco #25200072), incubatedat 37ºC for 5 min, collected into a 1.5-mL tube, and centrifuged at 1,000 rpm and room temperature for5 min, followed by resuspension to ~1 x 106 cells in 1x PBS containing 2% FBS in a 5-mL polystyrene
round-bottom tube (Falcon #352054). The cell suspension was immediately placed on ice until sorting.
The sorting was performed using MoFlo Astrios EQ Cell Sorter (Beckman Coulter). Cells were first gatedusing FSC-A and SSC-A, and the gate for EGFP+ cells was determined to obtain those having high
FITC-A intensities that were not observed in a non-transduction control cell sample. EGFP+ positive21

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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cells were single-cell sorted into 96-well plate wells, with the rest sorted in bulk to a single well of 96well plate, each with 100 µL of the mESC culture media. Approximately 100–1,000 EGFP+ cells wererecovered from each experiment, except for Clone 153, for which EGFP+ cells above the gating
threshold could not be observed.
Barcode sequencing library preparation
CloneSelect C→T plasmid library and barcoded cell population. To identify barcodes of the CloneSelectC→T plasmid library by high-throughput sequencing, ~10 ng of plasmid DNA (~1.0 x 109 molecules)
was used as a PCR template. To identify barcodes of the initial barcoded HEK293Ta cell population,
genomic DNA was purified using NucleoSpin Tissue (Macherey Nagel #740952) according to the
manufacturer’s protocol, and 119 ng of the extracted genomic DNA (4 x 104 molecules; 400-fold to theestimated barcode complexity) was used as a PCR template. The sequencing libraries were preparedby a two-step PCR method. The first-round PCR was performed in triplicate, each in 20 µL volume,
composed of template DNA, 0.5 µL each of 20 µM forward (SI#682) and reverse (SI#683) primers, 0.2µL of Phusion High-Fidelity DNA Polymerase (NEB #M0530), 4 µL of Phusion HF Buffer (NEB #B0518S),
2 µL of 2 mM dNTPs (Takara #4025), and 0.6 µL of 100% DMSO (NEB #12611P), with the following
thermal cycle condition: 98ºC for 10 s, 30 cycles of 98ºC for 10 s, 61ºC for 10 s, and 72ºC for 30 s, andthen 72ºC for 5 min for the final extension. Each PCR product was size-selected using 2% agarose gel,purified using PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). To provide Illumina sequencing
adapters and custom indices, the second-round PCR was performed to amplify each first-round PCR
replicate product in 20 µL volume, composed of 2.5 ng of the 1st PCR product, 1 µL each of 10 µM P5
and P7 custom index primers, 0.2 µL of Phusion High-Fidelity DNA Polymerase (NEB #M0530), 4 µL of
Phusion HF Buffer (NEB #B0518S), 2 µL of 2 mM dNTPs (Takara #4025), and 0.6 µL of 100% DMSO(NEB #12611P), with the following thermal cycle condition: 98ºC for 10 s, 20 cycles of 98ºC for 10 s,
61ºC for 10 s, and 72ºC for 30 s, and then 72ºC for 5 min for the final extension. Custom indices assigned
to the second-round PCR products can be found in Table S3. The second-round PCR products weresize-selected and purified using PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). The samples
were pooled, quantified by qPCR using KAPA Library Quantification Kit Illumina (KAPA BIOSYSTEMS#KK4824), and analyzed by paired-end sequencing using Illumina MiSeq.
Sorted HEK293T cells. For the amplicon sequencing-based identification of barcodes in low-volume
cells sorted after gRNA-dependent barcode-specific clone isolation using CloneSelect C→T, we
prepared a cell lysate for each sample as a PCR template. The sequencing library of each sample wasprepared by modifying the two-step PCR protocol described for purified DNA as a PCR template. Cells
in 8-strip PCR tubes were first incubated with 2.0 µL of lysis buffer, including 600 mM KOH, 10 mM
EDTA, and 100 mM DTT. The samples were then neutralized with 2.0 µL of neutralization buffer
composed of 0.4 µL of 1M Tris-HCl and 1.6 µL of 3 M HCl. The first-round PCR was performed byreplacing the template with 2.0 µL of the cell lysate. Although we did not observe visible gelelectrophoresis bands for the first-round PCR products, the PCR product of the expected size were sizeselected using 2% agarose gel, purified and eluted into 15 µL of ddH2O using PCR/Gel Extraction Kit(Nippon Genetics #FG-91302). The PCR products were quantified using Quant-iT PicoGreen dsDNA
Assay Kit (Thermo Fisher Scientific #P7589) and Infinite 200 PRO plate reader (TECAN) using Tecan
i-control software (version 1.10.4.0). The second-round PCR was performed for 2.0 ng of the first-round
PCR product. Custom indices assigned to the second-round PCR products can be found in Table S3.
The second-round PCR products were size-selected and purified using PCR/Gel Extraction Kit (Nippon
Genetics #FG-91302). The samples were pooled into a DNA LoBind 1.5-mL tube (Eppendorf #13-698791), quantified by qPCR using KAPA Library Quantification Kit Illumina (KAPA BIOSYSTEMS#KK4824), and analyzed by paired-end sequencing using Illumina MiSeq.
Uptag-dntag combination reference database. To identify the uptag-dntag combination reference
database of the mESC population tagged using the scCloneSelect barcode library, genomic DNA was
first extracted from 1 x 105 cells using NucleoSpin Tissue (MACHEREY-NAGEL #740952) according to
the manufacturer’s protocol. The sequencing libraries were prepared by a two-step PCR method. A total
22

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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of 50 ng genomic DNA was used for each PCR reaction. The first-round PCR was performed in duplicate,each in 20 µL volume, composed of template DNA, 0.7 µL each of 10 µM forward (SI#682) and reverse(RS#250) primers, 0.2 µL of Phusion High-Fidelity DNA Polymerase (NEB #M0530), 4.5 µL of Phusion
HF Buffer (NEB #B0518S), 1.6 µL of 2.5 mM dNTPs (NEB #N0447), with the following thermal cycle
condition: 98ºC for 10 s, 30 cycles of 98ºC for 10 s, 60ºC for 10 s, and 72ºC for 2 min, and then 72ºC
for 5 min for the final extension. Each PCR product was size-selected using 2% agarose gel, purified,and eluted into 20 µL of ddH2O using PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). To provide
Illumina sequencing adapters and custom indices, the second-round PCR was performed to amplify
each first-round PCR replicate product in 20 µL volume, composed of 20-fold dilution of the first-round
PCR product, 0.7 µL each of 10 µM P5 and P7 custom index primers, 0.2 µL of Phusion High-Fidelity
DNA Polymerase (NEB #M0530), 4.5 µL of Phusion HF Buffer (NEB #B0518S), 1.6 µL of 2.5 mM dNTPs(NEB #N0447), with the following thermal cycle condition: 98ºC for 10 s, 20 cycles of 98ºC for 10 s, 60ºC
for 10 s, and 72ºC for 30 s, and then 72ºC for 5 min for the final extension. Custom indices assigned tothe second-round PCR products can be found in Table S3. The second-round PCR products were sizeselected and purified using PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). The samples werepooled, quantified by qPCR using KAPA Library Quantification Kit Illumina (KAPA BIOSYSTEMS#KK4824), and analyzed by paired-end sequencing using Illumina MiSeq.
Sorted mESCs. For the amplicon sequencing-based identification of barcodes in cells sorted aftergRNA-dependent barcode-specific clone isolation using scCloneSelect, we prepared a cell lysate foreach sample as a PCR template. The sequencing library of each sample was prepared by modifying
the two-step PCR method described for the identification of the uptag-dntag combination reference
database. Sorted mESC samples were expanded in 96-well culture plate wells until confluent. Afteraspirating the culture medium, 20 µL of 50 mM NaOH was added to each well and transferred into a 96well PCR plate for direct cell lysis. The samples were then heated at 95ºC for 15 min, followed by cooling
down on ice. The samples were neutralized with 2.0 µL of 1 M Tris-HCl (pH 8.0). The first-round PCR
was performed in 40 µL volume replacing the template with 3.5 µL of cell lysate. The second-round PCR
was performed in 20 µL volume replacing the template with a 10-fold dilution of the first-round PCR
product. Custom indices assigned to the second-round PCR products can be found in Table S3. The
second-round PCR products were size-selected and purified using GeneJET Gel Extraction Kit (Thermo
Fisher Scientific #K0691). The samples were pooled into a DNA LoBind 1.5-mL tube (Eppendorf#0030108051), quantified by qPCR using KAPA Library Quantification Kit Illumina (KAPA
BIOSYSTEMS #KK4824), and analyzed by paired-end sequencing using Illumina HiSeq2500.
Reamplification of scCloneSelect dntags from Drop-seq library. To increase the sensitivity of identifying
dntags associated with single cells and their transcriptome profiles, we selectively reamplified the DNA
fragments encoding dntags and cell IDs from the intermediate Tn5 transposon-fragmented library of the
Drop-seq process and sequenced them separately. The reamplification PCR was performed in 20 µL
volume, composed of 1 ng of the cDNA library quantified using TapeStation with High Sensitivity D5000
ScreenTape (Agilent #5067-559 and #5067-5593), 0.7 µL each of 20 µM forward primer P5-TSO_Hybrid(45) and reverse primer SI#682, 0.2 µL of Phusion High-Fidelity DNA Polymerase (NEB #M0530), 4.5µL of 5x Phusion HF Buffer (NEB #B0518), and 1.6 µL of 2.5 mM dNTPs (NEB #N0447) with thefollowing thermal cycle condition: 95ºC for 30 s, 30 cycles of 98ºC for 30 s, 60ºC for 10 s, and 72ºC for
2 min, and 72ºC for 5 min for the final extension. The first-round PCR product was purified and eluted
into 20 µL of ddH2O using GeneJET Gel Extraction Kit (Thermo Fisher Scientific #K0691). The secondround PCR was then reamplified in 20 µL volume, composed of 10-fold dilution of the first-round PCR
product, 0.7 µL each of 10 µM P5 and P7 custom dual index primers, 0.2 µL of Phusion High-Fidelity
DNA Polymerase (NEB #M0530), 4.5 µL of 5x Phusion HF Buffer (NEB #B0518), 1.6 µL of 2.5 mM
dNTPs (NEB #N0447), with the following thermal cycle condition: 95ºC for 30 s, 15 cycles of 98ºC for
10 s, 65ºC for 10 s, and 72ºC for 2 min, and 72ºC for 5 min for the final extension. Custom indicesassigned to the second-round PCR products can be found in Table S3. The second-round PCR products
were size-selected using 2% agarose gel and purified using GeneJET Gel Extraction Kit (Thermo Fisher
Scientific #K0691). The samples were pooled, quantified by qPCR using KAPA Library Quantification23

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Kit Illumina (KAPA BIOSYSTEMS #KK4824), and analyzed by paired-end sequencing using Illumina
HiSeq2500.
Drop-seq
scRNA-seq was performed by Drop-seq. The Drop-seq platform was set up using devices manufactured
by Dolomite Bio following the manufacturer’s protocol. The microfluidic devices were fabricated by
YODAKA Co., Ltd. Cell samples were prepared at a concentration of ~2 x 105 cells/mL for analysis.
Sequencing libraries were prepared following the original Drop-seq paper (45). In brief, following
emulsion breakage and reverse transcription, “single-cell transcriptomes attached to microparticles”(STAMPs) were washed and proceeded to the Exonuclease I (NEB #M0293L) treatment. A total of~2,000 STAMPs were used for the whole cDNA amplification of each sample. After second-strand
synthesis, the library DNA was purified using AMPure XP beads (Beckman Coulter #A63881), quantified
using TapeStation with High Sensitivity D5000 ScreenTape (Agilent #5067-5592 and #5067-5593), andfragmented by Tn5 transposon using Nextera XT DNA Library Preparation Kit (Illumina #FC-131-1024)
according to the manufacturer’s protocol. The tagmented sequencing library was then purified using
AMPure XP beads (Beckman Coulter #A63881) and quantified by TapeStation with High Sensitivity
D5000 ScreenTape (Agilent #5067-5592 and #5067-5593). We confirmed each average library size tobe ~500 bp. Multiple scRNA-seq libraries were pooled and subjected to high-throughput sequencing
using Illumina MiSeq or HiSeq2500. The sequencing library index information can be found in Table S3.
mESC differentiation assay
For the establishment of a barcoded mESC population, cells were seeded on 6-well cell culture plate
wells at a density of ~2 x 105 cells/well with 2 mL of the culture medium containing 103 units/mL ESGRO
Recombinant Mouse Leukemia inhibitory factor (LIF) (Millipore #ESG1107) and 2i (1.0 µM PD0325901
Toris #4423 and 3.0 µM CHIR99021 Wako #038-23101). The next day, cells were transduced with 500µL of 15-fold concentrated barcoded lentivirus pool of scCloneSelect. Two days after, the culture
medium was exchanged with a fresh medium containing 1.0 µg/mL of Puromycin (Gibco #A1113803).
To restrict the clone complexity for the downstream cell differentiation and clone isolation assays, aclonal population bottleneck was created where ~1,000 cells were seeded on a 6-well cell culture plate
well and cultured for ten days. Cells were then split as follows: ~1 x 104 cells were seeded into theculture medium with LIF and 2i (LIF+2i+), ~1 x 104 cells were seeded into the culture medium without
LIF or 2i (LIF–2i–), two samples of ~1 x 105 cells each were used for the identification of uptag-dntag
combination reference database, and five replicates of ~1 x 105 cells were stored at –80ºC using
CELLBANKER 1 freeze media (ZENOAQ #11910). Four days later, the cell cultures of the LIF+2i+ and
LIF–2i– conditions were subjected to scRNA-seq.
RT-PCR-based analysis of barcode transcription
The transcription of polyadenylated scCloneSelect barcode products was analyzed by RT-PCR and gelelectrophoresis. Total RNA was first extracted using ISOSPIN Cell & Tissue RNA Kit (Nippon Gene
#314-08211) according to the manufacturer’s protocol. The RNA sample was then subjected to DNase
I treatment (Takara #2270B) to remove DNA and purified again using ISOSPIN Cell & Tissue RNA Kit(Nippon Gene #314-08211). The first-strand cDNA was synthesized using High-Capacity cDNA Reverse
Transcription Kit (Applied Biosciences #4368814). The reaction was performed in 10 µL volume,
composed of 5 µL of DNase I treated RNA (~1 µg), 0.5 µL of 100 µM oligo dT primer SI#4, 0.5 µL of
MultiScribe Reverse Transcriptase, 1 µL of 10x RT buffer, 0.4 µL of 100 mM dNTP, and 0.5 µL of RNAse
Inhibitor (Applied Biosciences #N8080119), with the following thermal cycler condition: 25ºC for 10 min,
37ºC for 12 min, and 85ºC for 5 min. Lastly, the transcription of the target barcode was tested by PCR
along with a GAPDH control. The PCR was performed in 20 µL volume, composed of 2 µL of 50-folddiluted first-strand cDNA, 2.8 µL total of a primer pair SI#116–SI#7 to amplify the dntag or a primer pair
RS#507–RS#508 to amplify GAPDH, 0.2 µL of Phusion DNA Polymerase (NEB #M0530S), 4 µL of 5x
Phusion HF Buffer (NEB #B0518), and 1.6 µL of 2.5 mM dNTPs (NEB #N0447), with the following
thermal cycler condition: 98ºC for 30 s, 30 cycles of 98ºC for 10 s, 60ºC for 10 s, and 72ºC for 30 s, and24bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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then 72ºC for 5 min for the final extension. The resulting PCR products were analyzed with 2% agarose
gel.

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Strains
Saccharomyces cerevisiae BY4741 (MATa his3∆0 leu2∆0 met15∆0 ura3∆0) was used for theexperiments using yeast CloneSelect system.

Yeast CloneSelectYeast CloneSelect library
To generate the yeast CloneSelect barcode library, a semi-random oligo pool KI#200 encoding 5′CCGWSNSWSNSWSNSWSNSNGTG-3′ was first chemically synthesized (Table S2) and amplified by
PCR in 40 µL volume, composed of 2 µL of 0.01 µM template, 2 µL each of 10 µM forward primer SI#368
and 10 µM reverse primer SI#369, 0.8 µL of Phusion High-fidelity DNA Polymerase (NEB #M0530S), 8µL of 5x Phusion HF Buffer (NEB #B0518S), and 2 µL of 2 mM dNTPs, with the following thermal cycle
condition: 98ºC for 30 s, 35 cycles of 98ºC for 10 s, 68ºC for 20 s, and 72ºC for 5 s, and then 72ºC for
5 min for the final extension. The PCR product was analyzed with 2% agarose gel and size-selected
and purified using FastGene PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). The purified
barcode fragment was then assembled into the cloning backbone plasmid pKI110 by Golden Gate
Assembly (66) using BsmBI (NEB #R0580S). We performed two assembly reactions, each in 25 µL
volume, composed of 500 fmol barcode fragments, 50 fmol backbone plasmid, 0.5 µL of BsmBI (NEB
#R0580S), 0.5 µL of T4 DNA Ligase (Nippon Gene #317-00406), 2.5 µL of 10x T4 DNA Ligation
Reaction Buffer (NEB #B0202S), and 0.125 µL of 60 mg/mL BSA (NEB #B9001S) with the following
thermal cycle condition: 15 cycles of 37ºC for 5 min and 20ºC for 5 min, and then 55ºC for 30 min forcomplete backbone digestion. For the bacterial transformation, 5 µL of the assembly product was used
to transform 50 µL of DH5α chemically competent cells (NEB #C2987I) according to the manufacturer’s
high-efficiency transformation protocol. Following one-hour outgrowth in 1 mL of SOC medium (NEB
#B9020S) at 37ºC, cells were plated on a total of four LB agar plates containing 100 µg/mL Ampicillin(Wako #014-23302). The cell sample was also diluted and spread on the selective plates to estimate
the clone complexity. To check assembly efficiency, random clones were isolated to perform genotyping
PCR with a primer pair of KI#169 and KI#170, validating each clone to have an expected barcode insert.
To construct Pool-100, 100 colonies were isolated, dissolved in 80 µL each of LB medium containing
100 µg/mL Ampicillin (Wako #014-23302), combined 5 µL each, and cultured overnight at 37ºC. The
plasmid DNA was extracted using FastGene Plasmid Mini Kit (Nippon Genetics #FG-90502). Pool-1580
was constructed by scraping and harvesting colonies from a plate with colony forming units close to
1,000 by adding 1.5 mL of LB media containing 100 µg/mL Ampicillin (Wako #014-23302). The resulting
cell samples were centrifuged at 13,000 rpm for 2 min to discard supernatant. The plasmid DNA pools
were then purified from the collected cells using FastGene PCR/Gel Extraction Kit (Nippon Genetics#FG-91302).
Barcoding of cells and introduction of genome editing reagents
For the barcoding of cells and introduction of genome editing reagents by transformation, we used the
Frozen-EZ Yeast Transformation II kit (Zymo Research #T2001) with slight modifications. Cells werefirst pre-cultured in 5 mL of YPDA or SC-Dropout medium (complying with the auxotrophic requirement
for plasmid maintenance in the host cells) in a cell culture tube rotating overnight at 30ºC. The next day,cells were cultured in 5 mL of a fresh YPDA medium with a starting OD600 nm of 0.3 and incubated until
the OD600 nm reached 0.8–1.0. After making competent cells according to the manufacturer’s protocol,
plasmid DNA and 50 µL of competent cells were added into a 1.5-mL tube, mixed thoroughly with 500µL of EZ3 solution according to the manufacturer’s protocol, and incubated at 30ºC for an hour withrotation. The cell sample was centrifuged at 13,000 rpm for 2 min, and the supernatant was discarded,followed by the addition of 2.5 mL of YPDA medium for recovery. After two-hour outgrowth at 30ºC withrotation, cells were centrifuged to remove the medium and washed with 1,000 µL of 1x TE twice. Cells
were then spread on SC–Dropout agar plates and incubated for two–four days at 30ºC.
25

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Barcoding of cells. When the background BY4741 cells were transformed using the barcode plasmid
library containing HIS3 marker, YPDA medium was used for pre-culturing and SC–His+Ade plates forselecting transformants. For the pooled barcoding of cells, the reaction was scaled to transform 250 µL
of competent cells using 200 ng of plasmid in total, and colonies formed on the selective plates werepooled and collected by scraping with 3–4 mL of SC–His+Ade. For the barcoding of cells using a single
barcode plasmid clone, 200 ng of plasmid was used to transform 15 µL of competent cells.
Introduction of genome editing reagents. When cells harboring the barcode plasmid containing HIS3
marker were subjected to a clone isolation, cells were transformed twice, first with the constitutively
active Target-AID plasmid pKI086 containing LEU2 marker and next with the targeting gRNA expression
plasmid encoding URA3 marker. For the first transformation, we used SC–His+Ade medium for preculturing and SC–His–Leu+Ade plates for selecting transformants. For the second transformation, SC–
His–Leu+Ade medium for pre-culturing and SC–His–Leu–Ura+Ade for selecting transformants. Whenthe background BY4741 cells were transformed using one of the galactose-inducible Cas9-based
enzyme plasmids (Cas9, dCas9, dCas9-PmCDA1, dCas9-PmCDA1-UGI, nCas9, nCas9-PmCDA1, andnCas9-PmCDA1-UGI) containing LEU2 marker and a CAN1-targeting gRNA plasmid containing URA3
marker, we used YPDA medium for pre-culturing and SC–Leu–Ura+Ade plates for selecting
transformants. For the barcode-specific reporter activation in a complex barcoded population, thereaction was scaled to transform 250 µL of competent cells using 200 ng of the enzyme plasmid and
200 ng of the targeting gRNA plasmid in total. For the other small-scale transformations, 200 ng of theeffector plasmid and 200 ng of the targeting gRNA plasmid were used to transform 15 µL of competentcells.
Barcode sequencing library preparation
To identify barcodes of the yeast CloneSelect plasmids introduced to the cells by high-throughput
sequencing, we first extracted and purified the barcode plasmids. We first centrifuged cells at 15,000rpm for 3 min, followed by discarding the supernatant. The cell pellet was resuspended with 20 µL of
Zymolyase Buffer, containing 2.5 mg/mL Zymolyase (Zymo Research #E1005), 10 mM Sodium
phosphate, and 1.2 M Sorbitol, and 500 µL of Solution I Buffer (Supplied in Zymolyase #E1005),
containing 0.1 M EDTA and 1 M Sorbitol. The sample was incubated at 37ºC for one hour andcentrifuged at 15,000 rpm for 1 min, followed by discarding the supernatant. The cell lysate sample wasthen incubated with 250 µL of Solution II Buffer (Supplied in Zymolyase #E1005), containing 20 mM
EDTA and 50 mM Tris-HCl, and 1% SDS, at 65ºC for 30 min, followed by the addition of 100 µL of 5 M
potassium acetate. The sample was further incubated on ice for 30 min and centrifuged at 15,000 rpm
for 3 min. The supernatant was transferred into a 1.5-mL tube, and the plasmid DNA was precipitatedwith the addition of 400 µL of isopropanol, followed by cleanup with 400 µL of 70% ethanol. The resulting
DNA pellet was resuspended with 50 µL of ddH2O containing 10 µg/mL RNase and incubated at 65ºC
for 10 min. Next, the barcode sequencing libraries were prepared by a two-step PCR method. The firstround PCR was performed in triplicate, each in 40 µL volume, composed of 1.0 µg of template DNA, 1µL each of 10 µM forward primer KI#169 and 10 µM reverse primer KI#289, 0.4 µL of Phusion HighFidelity DNA Polymerase (NEB #M0530S), 8 µL of Phusion HF Buffer (NEB #B0518S), and 0.8 µL of
10 mM dNTPs (Takara #4030), with the following thermal cycle condition: 98ºC for 30 s, 20 cycles of
98ºC for 10 s, 61ºC for 20 s, and 72ºC for 25 s, and then 72ºC for 5 min for the final extension. Each
PCR product was size-selected using 2% agarose gel, purified, and eluted into 50 µL of ddH2O using
PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). To provide Illumina sequencing adapters andcustom indices, the second-round PCR was performed to amplify each first-round PCR replicate productin 40 µL volume, composed of 2 µL of the first PCR product, 1 µL each of 10 µM P5 and P7 custom
index primers, 0.4 µL of Phusion High-Fidelity DNA Polymerase (NEB #M0530S), 8 µL of 5x Phusion
HF Buffer (NEB #B0518S), and 0.8 µL of 10 mM dNTPs (Takara #4030), with the following thermal cycle
condition: 98ºC for 30 s, 15 cycles of 98ºC for 10 s and 60ºC for 10 s, and 72ºC for 1 min, and then 72ºC
for 5 min for the final extension. Custom indices assigned to the second-round PCR products can befound in Table S3. The second-round PCR products were size-selected and purified using PCR/Gel
26

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Extraction Kit (Nippon Genetics #FG-91302). The sequencing libraries were pooled, quantified by qPCR
using KAPA Library Quantification Kit Illumina (KAPA BIOSYSTEMS #KK4824), pooled with equimolar
ratios, and analyzed by paired-end sequencing using Illumina HiSeq2500.
Analysis of reporter activation efficiency
To analyze the efficiency of the gRNA-dependent barcode-specific mCherry reporter activation, we
treated three independent barcoded cell samples, each by all three corresponding gRNAs (3x3 assay).
Each sample was spread on SC–His–Leu–Ura+Ade agar plates, scraped and inoculated to a 1.5-mL
tube containing 500 µL of SC–His–Leu–Ura+Ade medium, and cultured for two–four days at 30ºC. The
20 µL of pre-cultured cell sample was mixed with 180 µL of SC–His–Leu–Ura+Ade media andtransferred into a flat-bottom transparent 96-well plate (Greiner Bio-One #655090). Samples were thenanalyzed using Infinite 200 PRO plate reader (TECAN) using Tecan i-control software (version 1.10.4.0)
to measure mCherry fluorescence intensities normalized by OD595 nm values. For microscopic
observation of cells, 2.5 µL of cell sample was transferred on a glass slide, gently covered with a glass
coverslip, and observed using BZ-X710 (Keyence) with 20x and 40x objective lenses. We also directly
measured the GTG→ATG conversion rate of each sample by high-throughput sequencing. Cells were
scraped and harvested from the selective plates and lysed by DNAZol (COSMO BIO #DN127) according
to the manufacturer’s protocol. The sequencing libraries were then prepared by a two-step PCR method.
The first-round PCR was performed in 32 µL volume, composed of 1.6 µL of cell lysate, 1.6 µL each of
10 µM forward primer KI#168 and 10 µM reverse primer KI#169, 0.64 µL of Phusion High-Fidelity DNA
Polymerase (NEB #M0530S), 6.4 µL of Phusion HF Buffer (NEB #B0518S), and 0.64 µL of 10 mM
dNTPs (Takara #4030), with the following thermal cycle condition: 98ºC for 30 s, 30 cycles of 98ºC for
10 s, 61ºC for 10 s, and 72ºC for 1 min, and then 72ºC for 5 min for the final extension. The remaining
procedure was processed by the same protocols described for the barcode sequencing library
preparation. Custom indices assigned to the second-round PCR products can be found in Table S3.
The second-round PCR products were size-selected and purified using PCR/Gel Extraction Kit (Nippon
Genetics #FG-91302). The samples were pooled, quantified by qPCR using KAPA Library Quantification
Kit Illumina (KAPA BIOSYSTEMS #KK4824), and analyzed by paired-end sequencing using Illumina
MiSeq.
Isolation and analysis of barcoded colonies
After the barcode-specific reporter activation of a complex population, cells from test and control
conditions were spread on SC–His–Leu–Ura+Ade agar plates and imaged under a blue light illuminator
FAS-IV (Nippon Genetics) to distinguish mCherry+ and mCherry– colonies. Colonies were isolated into
96-well cell culture plate wells containing 98 µL of SC–His–Leu–Ura+Ade and cultured overnight at 30ºC.
Samples were then analyzed using Infinite 200 PRO plate reader (TECAN) using Tecan i-control
software (version 1.10.4.0) to measure mCherry fluorescence intensities normalized by OD595 nm values.
The same colony isolates were also subjected to Sanger sequencing to identify their barcode sequences
and base editing outcomes. Using the same cell lysis, first-round PCR, and PCR cleanup protocols asin the analysis of the reporter activation efficiency, we obtained barcode DNA fragments from eachsample. Each PCR product was analyzed by Sanger sequencing using a sequencing primer SI#658.
The Sanger sequencing trace was analyzed using PySanger (https://github.com/ponnhide/PySanger).
Canavanine assay
Genome editing efficiencies of different Cas9-based genome editing enzymes (Cas9, dCas9, dCas9PmCDA1, dCas9-PmCDA1-UGI, nCas9, nCas9-PmCDA1 and nCas9-PmCDA1-UGI) were estimatedby Canavanine assay, where enzymes were introduced to the cells with a gRNA targeting an argininetransporter CAN1 and its knockout efficiency can be assessed by cell survival under the presence of a
toxic arginine analog Canavanine. In this assay, Cas9-based enzyme expressions were regulated undera galactose-inducible GAL1/10 promoter. To induce genome editing, cells containing enzyme and gRNA
plasmids were first cultured in SC–Leu–Ura medium containing 2% glucose at 30ºC until saturation.
Cells were then resuspended in SC–Leu–Ura medium containing 2% raffinose with 16-fold dilution andcultured at 30ºC until saturation. Finally, cells were resuspended in SC–Leu–Ura medium containing27

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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2% raffinose and 0.02% galactose with 32-fold dilution and cultured at 30ºC for two days. Each sample
was then spread on SC–Leu–Ura–Arg+Ade and SC–Leu–Ura–Arg+Ade containing 60 mg/ml
canavanine plates and cultured at 30ºC for two–four days to estimate colony forming units and perform
spot assays. Furthermore, we scraped and harvested colonies from the SC–Leu–Ura–Arg+Ade control
plates to extract genomic DNA samples for the mutation spectra assay by high-throughput sequencing.
We first lysed 20 µL of cells at OD600 nm of 1.0 using 100 µL of DNAzol (COSMO BIO #DN127) followed
by incubation at room temperature for 15 min. The lysate was mixed thoroughly with 30 µL of 1M NaCland 50 µL of 100% ethanol and centrifuged at 14,000 rpm for 10 min. The supernatant was discarded,
and the pellet was washed with 550 µL of 70% ethanol. After air-drying, the sample was resuspended
in 50 µL of ddH2O. We then prepared an amplicon sequencing library for each sample by a two-step
PCR method in triplicate. The first-round PCR was in 40 µL volume, composed of 2 µL of template DNA,
2 µL each of 10 µM forward primer #KN85F3 and 10 µM reverse primer #KN85R2, 0.8 µL of Phusion
High-Fidelity DNA Polymerase (NEB #M0530S), 8 µL of Phusion HF Buffer (NEB #B0518S), and 0.8µL of 10 mM dNTPs (Takara #4030), with the following thermal cycle condition: 98ºC for 30 s, 30 cyclesof 98ºC for 10 s, 60ºC for 10 s, and 72ºC for 1 min, and then 72ºC for 5 min for the final extension. We
also performed the same process using a primer pair #HO2F2–#HO2R2 to prepare control samples.
The remaining procedure was processed by the same protocols described for the barcode sequencing
library preparation. Custom indices assigned to the second-round PCR products can be found in Table
S3. The second-round PCR products were size-selected and purified using PCR/Gel Extraction Kit(Nippon Genetics #FG-91302). The sequencing libraries were pooled, quantified by qPCR using KAPA
Library Quantification Kit Illumina (KAPA BIOSYSTEMS #KK4824), pooled with equimolar ratios, andanalyzed by paired-end sequencing using Illumina MiSeq.

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Preparation of cells for various bacterial CloneSelect systems
We prepared cell samples harboring single barcode plasmids for different variants of bacterial
CloneSelect systems (Table S2). The plasmid for the EGFP reporter-based system was introduced toBL21(DE3) Competent E. coli cells (NEB #C2527I), and the plasmids for the Blasticidin and Zeocin
resistance marker-based systems were introduced to T7 Express chemically competent cells (NEB
#C2566I) according to the manufacturer’s high-efficiency transformation protocols. The transformants
were selected on LB agar plates containing 100 µg/mL Ampicillin (Wako #014-23302) and/or 50 µg/mL
Kanamycin (Wako #111-00344).

Bacterial CloneSelectBacterial CloneSelect library
To generate the bacterial CloneSelect barcode library for the Zeocin resistance marker, a semi-randomoligo pool KI#405 encoding 5′-ATGCCGVNNVNNVNNVNNVNNTAA-3′ was chemically synthesized(Table S2), where a start codon ATG, the antisense strand sequence of 5′-CGG-3′ PAM and a quintuple
repeat of VNN (V=non-T) were followed by a TAA stop codon. The VNN repeat restricts additional stopcodons from appearing in-frame to the downstream reporter. The semi-random oligo pool was amplified
by PCR in 20 µL volume, composed of 1 µL of 1 μM template, 1 µL each of 10 µM forward primer SI#368
and 10 µM reverse primer SI#369, 0.4 µL of Phusion High-fidelity DNA Polymerase (NEB #M0530L), 4µL of 5x Phusion HF Buffer (NEB #B0518S), and 0.4 µL of 10 mM dNTPs, with the following thermal
cycle condition: 98ºC for 30 s, 20 cycles of 98ºC for 10 s, 68ºC for 20 s, and 72ºC for 20 s, and then72ºC for 5 min for the final extension. The PCR product was analyzed with 2% agarose gel and sizeselected and purified using FastGene PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). The
purified barcode fragment was then assembled into the cloning backbone plasmid pKI243 by Golden
Gate Assembly using BsmBI (66). We performed the assembly in 12.5 µL volume, composed of 2.91
fmol of barcode fragments, 14.9 fmol of backbone plasmid, 0.25 µL of BsmBI (NEB #R0580L), 0.5 µL
of T4 DNA Ligase (Nippon Gene #317-00406), 1.25 µL of 10x T4 DNA Ligation Reaction Buffer (NEB
#B0202S), and 0.62 µL of 2 mg/mL BSA (NEB #B9001S) with the following thermal cycle condition: 15
cycles of 37ºC for 5 min and 20ºC for 5 min, and then 55ºC for 30 min for complete backbone digestion.
For the bacterial transformation, 3 µL of the assembly product was used to transform 65 µL of T7
28

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Express chemically competent cells (NEB #C2566I) according to the manufacturer’s high-efficiency
transformation protocol. Following one-hour outgrowth in 500 µL of SOC medium (NEB #B9020S) at
37ºC, the cell sample was plated 250 µL each on three LB agar plates containing 100 µg/mL Ampicillin(Wako #014-23302). The cell sample was also diluted and plated on the selective plates to estimate theclone complexity. To check assembly quality and efficiency, 12 random clones were isolated andsubjected to Sanger Sequencing, validating that 11 out of 12 clones showed an expected clone barcode
insert. To construct Pool-100, 100 colonies were isolated, dissolved in 80 µL each of LB medium
containing 100 µg/mL Ampicillin (Wako #014-23302), combined 5 µL each, and cultured overnight at
37ºC. The plasmid DNA was extracted using FastGene Plasmid Mini Kit (Nippon Genetics #FG-90502).
Pool-1550 was constructed by scraping and harvesting colonies from a plate with colony forming units
close to 1,000 by adding 1.5 mL of LB media containing 100 µg/mL Ampicillin (Wako #014-23302). The
barcode plasmid libraries were used to transform T7 Express chemically competent cells (NEB #C2566I)
to establish barcoded E. coli cell populations.
Barcode sequencing library preparation
For each of the Pool-100 and Pool-1550 barcode plasmid libraries, the barcode sequencing library was
prepared in triplicate by a two-step PCR method. The first-round PCR was performed in five separate
PCR reactions, each in 40 µL volume, composed of 2.0 ng of plasmid template DNA, 1 µL each of 10µM forward primer KI#403 and 10 µM reverse primer KI#404, 0.4 µL of Phusion High-Fidelity DNA
Polymerase (NEB #M0530L), 8 µL of Phusion HF Buffer (NEB #B0518S), and 0.8 µL of 10 mM dNTPs(Takara #4030), with the following thermal cycle condition: 98ºC for 30 s, 20 cycles of 98ºC for 10 s,
54ºC for 20 s, and 72ºC for 25 s, and then 72ºC for 5 min for the final extension. For each replicate, thefive PCR reaction products were pooled, size-selected using 2% agarose gel, purified, and eluted into
30 µL of ddH2O using PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). To provide Illumina
sequencing adapters and custom indices, the second-round PCR was performed to amplify each firstround PCR replicate product in 40 µL volume, composed of 2 µL of the first PCR product, 1 µL each of
10 µM P5 and P7 custom index primers, 0.4 µL of Phusion High-Fidelity DNA Polymerase (NEB
#M0530L), 8 µL of 5x Phusion HF Buffer (NEB #B0518S), and 0.8 µL of 10 mM dNTPs (Takara #4030),
with the following thermal cycle condition: 98ºC for 30 s, 15 cycles of 98ºC for 10 s, 60ºC for 10 s, and72ºC for 60 s, and then 72ºC for 5 min for the final extension. Custom indices assigned to the secondround PCR products can be found in Table S3. The second-round PCR products were size-selected
and purified using PCR/Gel Extraction Kit (Nippon Genetics #FG-91302). The sequencing libraries werepooled, quantified by qPCR using KAPA Library Quantification Kit Illumina (KAPA BIOSYSTEMS#KK4824), pooled with equimolar ratios, and analyzed by paired-end sequencing using Illumina MiSeq.
Introduction of genome editing reagentsTo introduce a plasmid containing ABE-7.10 and gRNA with given induction systems to barcoded cells,
we used Mix&Go! E. coli Transformation Kit (Zymo Research #T3001) according to the manufacturer’s
protocol. To select successful transformants, the transformation reaction sample was plated on LB agar
plates containing 100 µg/mL Ampicillin (Wako #014-23302) and 50 µg/mL Kanamycin (Wako #11100344) and incubated overnight at 37ºC. For the experiments that involved Arabinose (Sigma-Aldrich#A3256-10MG) and Isopropylthio-β-galactoside (IPTG) (ThermoFisher Scientific #15529019)
inductions, cells were cultured in the medium containing 100 mM Arabinose and 0.1 mM IPTG overnightat 37ºC prior to the analyses. For the barcoded cell isolation with the Zeocin-resistance marker-based
system, we used a low-salt LB medium adjusted to pH 7.5 with 1 M NaOH (Nakalai #37421-05) for the
Zeocin activity. We also decided to use 100 µg/mL Zeocin (Invitrogen #R25001) or 100 µg/mL Blasticidin
S (Wako #029-18701) with no Arabinose or IPTG for genome editing and selecting reporter-activatedcells, as leaky gene editing reagent expressions from the inducible promoters with the no inducer
condition were sufficient and maximized the cell viability. The information on the genome editing
plasmids used in this study can be found in Table S2.

29

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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Analysis of reporter activation efficiency
To analyze the efficiency of the gRNA-dependent barcode-specific EGFP-reporter activation, 200 µL ofcell samples were transferred into a flat-bottom transparent 96-well plate (Greiner Bio-One #655090)
and analyzed using Infinite 200 PRO plate reader (TECAN) using Tecan i-control software (version
1.10.4.0) to measure EGFP fluorescence intensities normalized by OD595 nm values. For microscopic
observation of cells, 2.5 µL of cell sample was transferred onto a glass slide (MATSUNAMI #S2441),
gently covered with a glass coverslip, and observed using BZ-X710 (Keyence) with 20x and 40x
objective lenses.

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High-throughput sequencing
The sequencing libraries were mixed with 20–30% of PhiX spike-in DNA control (Illumina #FC-1103001) for better cluster generation on the flow cell and sequenced by Illumina MiSeq (MiSeq v3 150cycles kit #MS-102-3001 or 300-cycles kit #MS-102-3003), HiSeq2500 (TruSeq rapid SBS kit v2 #FC402-4022). Base calling was performed with bcl2fastq2 (version v2.20.0) to generate FASTQ files. The
sequencing condition for each library and the NCBI Sequence Read Archive’s ID to each raw FASTQ
file can be found in Table S3.

Isolation and analysis of barcoded colonies
After the barcode-specific Zeocin-resistance marker activation in the barcoded cell population, barcodesof colonies from test and control conditions were analyzed by Sanger sequencing. For each colony, thebarcode region was amplified by PCR in 20 µL volume, composed of 1 µL of cell suspension, 0.5 µL
each of 10 µM forward primer KI#403 and 10 µM reverse primer KI#404, 0.2 µL of Phusion High-Fidelity
DNA Polymerase (NEB #M0530L), 4 µL of Phusion HF Buffer (NEB #B0518S), and 0.4 µL of 10 mM
dNTPs (Takara #4030), with the following thermal cycle condition: 98ºC for 30 s, 30 cycles of 98ºC for
10 s, 54ºC for 20 s, and 72ºC for 30 s, and then 72ºC for 5 min for the final extension. The PCR products
were analyzed with 2% agarose gel and transferred into 96-well PCR plate wells for clean-up with 20µL of AMPure XP beads (Beckman Coulter #A63881) according to the manufacturer’s protocol. The
Sanger sequencing was performed using a sequencing primer KI#403. The Sanger sequencing trace
was analyzed using PySanger (https://github.com/ponnhide/PySanger).
High-throughput sequencing and data analysis

Barcode identification
Mammalian CloneSelect C→T and yeast and bacterial CloneSelect. Sequencing reads were aligned tothe constant sequences of the designed library structure using NCBI BLAST+ (version 2.6.0) (67) withthe blastn-short option to identify sample indices for demultiplexing and CloneSelect barcode sequences.
To generate the barcode allowlist for the mammalian CloneSelect C→T mini-pool experiments, barcode
sequences commonly identified in both the plasmid DNA library and genomic DNA library were firstobtained. Their sequencing errors were then corrected using starcode (version 1.4)
(https://github.com/gui11aume/starcode) with the maximum Levenshtein distance threshold of 4 tomerge minor and major barcodes. The barcode counts of each sample were normalized by the total
barcode count. To estimate barcode frequencies for a given cell or DNA pool sample, the barcode
frequencies were averaged across replicates if any. The barcode sequence and frequency information
obtained in this study can be found in Table S1. Codes are available athttps://github.com/yachielab/CloneSelect_v1/tree/main/Barcode_identification/CloneSelect.
scCloneSelect. To identify uptag and dntag barcodes of the barcoded cells, we first demultiplexed thesequencing reads and used cutadapt v4.1 (https://github.com/marcelm/cutadapt/) to extract uptag anddntag sequences sandwiched between their 20-bp upstream and downstream constant sequences. The
identified uptags and dntags were filtered with the Q-score threshold of 30, clustered, and further filteredwith their lengths of 17-bp for uptags and 30-bp for dntags using bartender-1.1(https://github.com/LaoZZZZZ/bartender-1.1) (68). For the construction of the uptag-dntag combinationreference database, uptags and dntags found in the same reads were first paired. We discarded30

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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redundant uptag-dntag pairs whose uptag or dntag were found in more abundant pairs. For the mapping
of
dntag
to
the
uptag-dntag
database,
we
used
symspellpy
v6.7(https://github.com/mammothb/symspellpy) to find one with the shortest edit distance. In cases wheremultiple dntags in the database were found to be mapped by a query with the same edit distances, thedntag with the highest frequency in the database was chosen. To analyze uptag frequencies in a cell
population obtained after gRNA-dependent barcode-specific EGFP reporter activation followed by flow
cytometry cell sorting, we mapped the sequencing reads to the uptag-dntag database and obtained readcounts for uptags recorded in the database using bartender-1.1 and symspellpy v6.7. Conflicts by the
redundant hits to the database were resolved in the same way as those for dntags described above.
Codes are available athttps://github.com/yachielab/CloneSelect_v1/tree/main/Barcode_identification/scCloneSelect.

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The statistical tests were performed using R (version 4.2.0). The details of each statistical test can befound in the corresponding figure legend. The statistical methods used in this study and P-values arealso listed in Table S4.

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High-throughput sequencing data generated in this study are available at the NCBI Sequence Read
Archive (PRJNA901977). All the codes used in this study are available athttps://github.com/yachielab/CloneSelect_v1.

Drop-seq data analysis
After sample demultiplexing, FASTQ files were processed with Dropseq Tools v2.5.1(https://github.com/broadinstitute/Drop-seq) for base quality filtering, adapter trimming, and Cell ID and
UMI extraction. Picard v2.18.14 (https://github.com/broadinstitute/picard) was used to convert BAM files
to FASTQ files for the proceeding step. The filtered reads were aligned using STAR v2.7
(https://github.com/alexdobin/STAR) (69) using the mm10 reference genome. The differential gene
expression and clustering analysis were performed using Seurat v3 (https://github.com/satijalab/seurat)(70). After filtering cells with the thresholds of Feature_RNA > 200 & nFeature_RNA < 2500 & percent.mt< 5, their gene expression profiles were normalized before clustering with the Seurat::sctransformfunction. The dntags to be analyzed in this study were first determined in the first Drop-seq run basedon their cumulative read count distribution of Cell IDs with the threshold determined by the knee point
using the Python package kneed v0.8.1 (https://github.com/arvkevi/kneed). However, to map dntag
distribution to the single-cell transcriptome data with high sensitivity, we also sequenced the dntagreamplified library and identified the association of Cell IDs and dntags in the dntag-uptag combinationreference database by the method described above for the scCloneSelect uptag and dntag identification.
In cases where multiple dntags were found for a single Cell ID, the dntag with the most abundant UMI
count was chosen. Codes are available athttps://github.com/yachielab/CloneSelect_v1/tree/main/Drop-seq.
Mutational spectra analysis
The amplicon sequencing reads obtained to analyze mutational patterns at the CAN1 target site
conferred by each of the Cas9-based genome editing enzymes was processed by a pipeline described
previously (39). Codes specific to this study are available athttps://github.com/yachielab/CloneSelect_v1/tree/main/Mutational_Spectra_Analysis.
Statistical analysis

Data and code availability

31bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Supplementary Materials for

A multi-kingdom genetic barcoding system for
precise target clone isolation
Soh Ishiguro1,15,19, Kana Ishida2,19, Rina C. Sakata1,19, Hideto Mori3-5, Mamoru Takana3,
Samuel King1, Omar Bashth1, Minori Ichiraku6, Nanami Masuyama1,4,5, Ren Takimoto1, Yusuke
Kijima1,7, Arman Adel1, Hiromi Toyoshima3, Motoaki Seki3, Ju Hee Oh8, Anne-Sophie
Archambault8, Keiji Nishida9,10, Akihiko Kondo9-11, Satoru Kuhara12, Hiroyuki Aburatani13,
Ramon I. Klein Geltink8, Yasuhiro Takashima6, Nika Shakiba1, and Nozomu Yachie1,3,14,16-18*
1.

School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The
University of British Columbia, Vancouver, Canada.
2. Spiber Inc., Tsuruoka, Japan.
3. Synthetic Biology Division, Research Center for Advanced Science and Technology, The
University of Tokyo, Tokyo, Japan.
4. Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
5. Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa,
Japan.
6. Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
7. Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Tokyo, Japan.
8. BC Children’s Hospital Research Institute, Department of Pathology and Laboratory Medicine,
The University of British Columbia, Vancouver, Canada.
9. Engineering Biology Research Center, Kobe University, Kobe, Japan.
10. Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
11. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe
University, Kobe, Japan.
12. Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu
University, Fukuoka, Japan.
13. Genome Science Division, Research Center for Advanced Science and Technology, The
University of Tokyo, Tokyo, Japan.
14. Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University,
Suita, Osaka, Japan
15. Twitter: @soh__i
16. Twitter: @nzmyachie
17. Twitter: @yachielab
18. Mastodon: sciencemastodon.com/@nzm
19. These authors contributed equally.
*Corresponding author: nozomu.yachie@ubc.ca.

1bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Table of Contents
Fig. S1
Fig. S2
Fig. S3
Fig. S4
Fig. S5
Fig. S6
Fig. S7
Fig. S8
Fig. S9
Fig. S10
Fig. S11
Fig. S12
Fig. S13
Fig. S14
Table S1
Table S2
Table S3
Table S4

Supplementary data for developing CloneSelect C→T
Supplementary data for comparing CloneSelect C→T with CRISPRa-based circuits
DSB-based circuit
mCherry reporter for CloneSelect C→T
Supplementary data for comparing CloneSelect A→G with CRISPRa-based circuits
Supplementary data for isolating barcoded cells using CloneSelect C→T
Supplementary data for developing scCloneSelectThe gene expression profile cytometry cell sorting concept
Supplementary data for isolating barcoded cells identified in scRNA-seq data
Supplementary data for developing yeast CloneSelectPreparation of yeast CloneSelect library
Supplementary data for developing bacterial CloneSelectPreparation of E. coli CloneSelect libraryMultiple gRNA-input logic gates
Barcode count distributions observed in this study
Oligonucleotides and plasmids used in this study
List of high-throughput sequencing datasets
Statistical tests performed in this study and P-values2bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S1. Supplementary data for developing CloneSelect C→T. (A–C) Barcode-specific gRNA-dependent
activation of EGFP reporters for two barcoded HEK293T strains established for each of CloneSelect C→T (A),low-copy CRISPRa (B), and high-copy CRISPRa (C) (n=3). Scale bar, 50 µm. Welch’s t-test was performed tocompare on-target (OT) and non-target (NT) activations. (D) Median EGFP intensities of genome editing-activated
EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. (E) Comparison of
Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set ofbarcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. (F) Reporter
activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT
activations. Scale bar, 80 µm. *P < 0.05; **P < 0.01; ***P < 0.001.

3

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S2. Supplementary data for comparing CloneSelect C→T with CRISPRa-based circuits. (A and B)
Barcode-specific gRNA-dependent reporter activation of six barcoded cell lines prepared for each of low-copy
CRISPRa and high-copy CRISPRa. Scale bar, 50 µm. (C) Median EGFP intensities of genome editing-activated
EGFP positive cells (n=3). (D) ROC curves along varying reporter intensity thresholds for target barcoded cells.
CloneSelect C→T, low-copy CRISPRa, and high-copy CRISPRa were examined for the common set of six
barcodes. The Mann-Whitney U test was performed to compare two groups (*P < 0.05; **P < 0.01; ***P < 0.001).
(E) Frequencies of CloneSelect C→T reporter-activated cells obtained by transfection of different DNA amountsof barcode-targeting genome editing reagents. (F) ROC curve for each input DNA amount along varying reporter
intensity thresholds for target barcoded cells.

4bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S3. DSB-based circuit. (A) Barcode-specific gRNA-dependent reporter activation circuit using wild-type
Cas9. (B) Barcode-specific activation of the deletion-based reporter prepared for two barcodes (BC-del1 and BCdel2) in HEK293T cells (n=3). Welch’s t-test was performed to compare on-target (OT) and non-target (NT)
activations (*P < 0.05; **P < 0.01; ***P < 0.001). Scale bar, 50 µm. (C) ROC curves along varying reporter intensity
thresholds for target barcoded cells (n=3).

5

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S4. mCherry reporter for CloneSelect C→T. (A) Different mCherry reporter variants tested to establish
CloneSelect C→T. (B and C) mCherry expression from the different reporter variants with the first codon as GTG
or ATG. Scale bar, 50 µm. (D) Activation of the M1V (GTG)+∆2-9 mutant reporter with OT and NT gRNAs (n=3).
Scale bar, 100 µm.

6bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S5. Supplementary data for comparing CloneSelect A→G with CRISPRa-based circuits. (A–C) Barcodespecific gRNA-dependent reporter activation of three barcoded cell lines prepared for each of CloneSelect A→G,
low-copy CRISPRa, and high-copy CRISPRa. Scale bar, 50 µm. (D–F) Flow cytometry analysis of single-cell
EGFP activation levels. (G) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The
Mann-Whitney U test was performed to compare two groups (*P < 0.05; **P < 0.01; ***P < 0.001). (H) ROC curves
along varying reporter intensity thresholds for target barcoded cells (n=3).

7

bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S6. Supplementary data for isolating barcoded cells using CloneSelect C→T. (A) Schematic diagram
for the barcode library construction. The ∆EGFP fragment (no start codon) was amplified by PCR using pooled
forward primers encoding the PAM followed by semi-random barcode sequences encoding GTG and a common
reverse primer, and enzymatically digested and ligated to the lentiviral backbone. (B) The insert PCR fragments.
(C) Estimated complexities of the generated plasmid pools in triplicate. (D) Library QC by colony isolation andrestriction digest by BsrGI, ClaI and PvuI. (E) Sanger sequencing of the barcode region of the colony isolates. (Fand G) Barcode distribution in the lentiviral plasmid DNA pool and that in the cell population transduced using thesame plasmid DNA pool. (H) Barcode distribution in the EGFP-positive cell population obtained by cell sorting
after barcode-specific activation by a target gRNA. The results of the 16 independent samples were combined
after read count normalization for each sample. (I) Frequency of the GTG→ATG mutation observed for eachbarcode after sorting of the reporter positive cells. Each row represents the GTG→ATG mutation frequency profile
obtained for each target isolation attempt.

8bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S7. Supplementary data for developing scCloneSelect. (A) EGFP-positive control expressions for theoriginal CloneSelect C→T and scCloneSelect in HEK293T cells with the same genome editing conditions tested
for the respective reporters (n=3). Scale bar, 50 µm. (B) Median EGFP intensities of base editing-activated EGFP
positive cells (n=3). (C and D) Barcode-specific gRNA-dependent reporter activation of six barcoded cell lines by
scCloneSelect (n=1). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (*P< 0.05; **P < 0.01; ***P < 0.001). Scale bar, 50 µm. (E) RT-PCR of the scCloneSelect dntags in HEK293T. (F)Fraction of mESC single-cell transcriptome profiles (Drop-seq) that contained dntags and fraction of dntags
reported in the uptag-dntag combination reference database. (G) Schematic representation of a scCloneSelectreporter activation assay where Target-AID was stably introduced to the cell population prior to barcoding andgRNA-dependent reporter activation. (H and I) gRNA-dependent reporter activation of target barcoded mESCs
and CA1 hPSCs by scCloneSelect (n=2). Target-AID was stably integrated prior to the barcoding. Targeting9bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

gRNAs were delivered by transfection. Welch’s t-test was performed to compare OT and NT activations (*P < 0.05;**P < 0.01; ***P < 0.001). Scale bar, 100 µm. (J) Schematic representation of a scCloneSelect reporter activation
assay where the target gRNA and Target-AID were electroporated together to the barcoded cell population. (K)gRNA-dependent reporter activation of barcoded H1 hPSCs by scCloneSelect (n=2). Targeting gRNA and TargetAID were electroporated together. Scale bar, 100 µm.

10bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S8. The gene expression profile cytometry cell sorting concept. (A) Schematic diagram of a
scCloneSelect workflow. (B) Preparation of barcode library for scCloneSelect. (C) Sanger sequencing results of
the uptag and dntag regions of the constructed plasmid pool. (D) Mixing of genomic DNA samples from twobarcoded cell lines to optimize the PCR protocol to create a library for identifying the uptag-dntag combinationreference database. (E) Fraction of unexpected chimeric PCR recombination products in the sequencing reads.

11bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S9. Supplementary data for isolating barcoded cells identified in scRNA-seq data. (A) QC of scRNAseq datasets obtained for the barcoded mESC population cultured with LIF and 2i and that cultured without LIF or
2i. (B) Single-cell expression patterns of key genes. (C) Distribution of cells in a two-dimensional UMAP space forall the clones targeted for isolation. (D) Abundances of barcoded cell clones in the mESC population. The data
was generated based on dntags identified in the scRNA-seq dataset with no reamplification of the dntag reads.
(E) gRNA-dependent activation of the reporter for each of the target clones in the initial mESC population. (F)Culturing of single isolated cells. Scale bar, 100 µm. (G) Sanger sequencing of the barcodes for the isolated target
clones expanded after clone labeling and single-cell isolation.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S10. Supplementary data for developing yeast CloneSelect. (A) Different mCherry reporter variants tested
to establish CloneSelect C→T. The different reporter variants were tested with the first codon as GTG or ATG.
Scale bar, 100 µm. (B) Canavanine resistance assays for different CRISPR genome editing enzymes with a gRNAtargeting CAN1 gene and a control NT gRNA. For each experiment, cell concentration was normalized to 1.0
OD595 nm and serially diluted with 10-fold increments for spotting. (C) Estimated CFU counts for the same assay in(B). (D) Genome editing outcomes observed by amplicon sequencing. Frequencies of mutation patterns observed
across the target sequence region are shown for the same assay in (B). (E) Genome editing frequencies at thetarget CAN1 locus estimated by amplicon sequencing for the different enzymes. (F) Activation of the mCherry
M1V (GTG)+∆2-9 mutant reporter by OT and NT gRNAs. Scale bar, 200 µm. (G) mCherry-positive control

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

expressions for yeast CloneSelect. Yeast cells having the positive control reporters with three different barcodes(BC-C1, BC-C2, and BC-C3) were each treated by Target-AID and three different targeting gRNAs. Scale bar, 25µm.

14bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S11. Preparation of yeast CloneSelect library. (A) Schematic diagram for the barcode library construction.
The barcode fragment pool was prepared by PCR using a common primer pair amplifying a template DNA poolencoding the PAM, WSNS semi-random repeat, and the mutated start codon GTG. The PCR product was digested
and ligated to a backbone plasmid. (B) The insert PCR fragments (Pool-100). (C) Library QC by colony isolation
and PCR amplification of the barcode insert (Pool-100). (D) Barcode abundance distribution (read per million) in
the constructed barcode library pool. The sequencing library was prepared and analyzed in triplicate (n=3).

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S12. Supplementary data for developing bacterial CloneSelect. (A) Activities of the positive control EGFP
reporter. ABE and gRNA expression were controlled by an IPTG-inducible promoter, and the EGFP reporter
expression was controlled by an arabinose-inducible promoter. (B) Base editing outcomes of the positive control
reporters analyzed by Sanger sequencing. (C) Testing of Zeocin resistances conferred by two promotersexpressing a Zeocin resistance gene with and without the upstream stop codon to block the selective marker
translation. Each cell sample concentration was first adjusted to 0.1 OD595 nm and serially diluted with 10-foldincrements for spotting 5 µL. (D) Testing of cell viability under a non-selective condition for a constitutively active
T7 promoter and the IPTG-inducible promoter to express the gRNA. OT and NT gRNAs were tested for the gRNAdependent EGFP reporter and the positive control EGFP reporter. ABE was expressed under the IPTG-inducible
promoter without IPTG provided. (E) gRNA-dependent Zeocin resistance reporter activation tested for the IPTGinducible promoters with and without IPTG. (F) Bacterial CloneSelect using the Blasticidin resistance gene. Eachcell sample concentration was first adjusted to 0.1 OD595 nm and serially diluted with 10-fold increments for spotting
5 µL. (G) gRNA-dependent Blasticidin-resistance reporter activation tested for different inducer conditions anddifferent Blasticidin concentrations. Each cell sample concentration was adjusted to 0.1 OD595 nm for spotting 5 µL.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S13. Preparation of E. coli CloneSelect library. (A) Schematic diagram for the barcode library construction.
The barcode fragment pool was prepared by PCR using a common primer pair amplifying a template DNA poolencoding the PAM, VNN repeat sequence, and the start codon TAA. The PCR product was digested and ligatedto a backbone plasmid. (B) Barcode abundance distribution (read per million) in the constructed barcode librarypool. The sequencing library was prepared and analyzed in triplicate (n=3).

17bioRxiv preprint doi: https://doi.org/10.1101/2023.01.18.524633; this version posted January 24, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeavailable under aCC-BY-NC-ND 4.0 International license.

Fig. S14. Multiple gRNA-input logic gates. (A) Three-gRNA-input OR gate with CloneSelect C→T that isdesigned to confer the EGFP reporter expression by any of the three barcode-specific gRNA-dependent
GTG→ATG mutations. (B) Three-gRNA-input AND gate with CloneSelect A→G that is designed to confer the
EGFP reporter expression when all three barcode-specific gRNA-dependent TAA→CAA mutations are provided.

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