description

Background

The fidelity of genomic sequence is the underpinning aspect of normal cellular activities. Even a minor
disruption to DNA, the storage center of genetic information, can lead to detrimental consequences,
including excessive cell death, abnormal proliferation, untimely cell senescence, etc. For example,
genomic instability and mutation is considered as one of the ten hallmarks of cancer, the occurrence
of which generally leads to over-activation of signaling pathways, in turn triggering malignant
proliferation of cells^[1]. Recurrent hot-spot DNA mutations and chromosomal rearrangements are
commonly observed in cancer samples. Genomic mutation is therefore a primary concern in terms of both
basic research and clinical treatment, demanding multi-disciplinary efforts.

In order to address this imperative issue, a gene mutation surveillance system that can sensitively and
efficiently detect and remove such aberrations to achieve real-time monitoring in vivo is required.
Essentially by designing such a system, a tool that can recognize aberrant mutation on mRNA sequences,
or more generally at the transcriptomics level, is desired, because transcripts are derived from
genomic sequences and hence are representatives of genomic instability and mutations. More importantly,
their availability and abundance in the cytoplasm allow them to be targeted. Nonetheless, few such
satisfying molecular tools have been developed yet. Consequently, to design such a surveillance
system is not only important but also necessary.

To serve such purpose, an RNA binding factor with high efficiency and sensitivity is a must, which
is able to distinguish RNA transcripts produced by mutated genes and switch on the suicidal gene
expression afterwards. There are three major RNA targeting methods currently: PUF proteins^[2][3][4],
molecular beacons^[5] and RNA aptamers^[6]. Due to lack of specificity or stability in vivo, those existing
methods are not suitable for the potential application in our gene mutation surveillance system.
However, it is newly reported that dCas9^[7], capable of recognizing specific endogenous mRNA
sequences, turns out to be an ideal candidate. Surveillance Cas9 (suvCas9), termed for the first time
by our team, is a variant of Cas9 without DNA cleavage activity and can target specific mRNA sequences
by manually designed sgRNA and PAM sequences. With this remarkable ability to target mRNA, suvCas9
can hopefully be adopted into our surveillance system as the monitor as well as the executor

We propose to construct suvCas9 system in Saccharomyces cerevisiae to function as the monitor
of genomic sequence. Saccharomyces cerevisiae is one of the most commonly used model organisms,
representing a simple eukaryote with a highly manipulatable genome. Apart from many physical properties
and technical advantages, such as rapid growth, minimal pathogenicity, the ease of phenotype identification,
a well-defined genetic system, and most importantly, a highly versatile DNA transformation system^[11],
the yeast has been reported to share a similar characteristics of in vivo ssDNA generation by reverse
transcriptase with prokaryotes^[9], which will guide suvCas9 to the targeted mRNA.

Project Overview

Genomic stability and DNA sequence fidelity are critical for normal cellular activities. If such storage
center of genetic information is disrupted, detrimental consequences will take place, including but not
limited to excessive cell death, abnormal proliferation, untimely cell senescence, etc. Therefore, a gene
mutation surveillance system that can sensitively and efficiently detect and remove such aberrations
in vivois required.


Saccharomyces cerevisiae is chosen as the model organism for designing such a gene surveillance system.
As one of the most commonly used tools, it not only has a highly manipulatable genome, rapid growth
speed, minimal pathogenicity, an ensemble of selectable markers, a well-defined genetic system, and most
importantly, a highly versatile DNA transformation system.

Essentially by designing such a system, a tool that can recognize aberrant mutation on mRNA sequences, or
more generally at the transcriptomics level, is desired, because transcripts are derived from genomic
sequences and hence are representatives of genomic instability and mutations. Surveillance Cas9 (suvCas9),
termed for the first time by our team, is a variant of Cas9 without DNA cleavage activity and can target
specific mRNA sequences by manually designed sgRNA and PAM sequences.

That being said, after mutation is detected on mRNAs, how can our system respond? In a nutshell, when
mutations occur, the surveillance system will detect and trigger the suicidal system. The surveillance
system can be further quantitatively optimized to maximize its sensitivity and minimize potential side
effects.

design

This project aims to establish a well-controlled gene mutation surveillance system to monitor
possible harmful gene mutations. When mutations occur, the surveillance system will detect and
trigger the suicidal system. The surveillance system can be further quantitatively optimized to
maximize its sensitivity and minimize potential side effects.

RNA targeting by CRISPR/Cas9 system

To achieve the surveilling functions of the whole circuit, a suvCas9 is facilitated by an sgRNA
and a deoxynucleotide oligonucleotide (PAMmer) that hybridizes to the target RNA, providing
the PAM sequence^[12]. suvCas9 plays an indispensable part in the coupling of mRNA recognition
system and the suicidal gene expression. suvCas9 performs high-fidelity mRNA binding activity,
of which the specificity is double-determined by sgRNA and PAMmer. This surveillance system is
composed of the dCas9 protein, sgRNA and PAMmer, forming a complex that can stably exist in the
cytoplasm and tightly bind to its targeted mRNA. When a mutation of the genes under surveillance
is detected by suvCas9, it can immediately trigger the suicidal system to eliminate the mutated
cell, avoiding further catastrophic consequences.

In vivo generation of ssDNA by SCRIBE system

In order to target mRNA in the cytoplasm, several concerns should be noticed^[7]. First, the PAM
motif, essentially an ssDNA in our project, should be provided together with sgRNA pairs, the
presence of which are prerequisites for suvCas9 to acquire its activity. Second, the genomic
counterpart of transcripts should be prevented from being recognized and disrupted by suvCas9,
otherwise the surveillance system will contradict the purpose of maintaining high genomic fidelity.
The newly developed RNA targeting system^[7] meets both criteria mentioned above: it creates the PAM motif
by introducing a special PAMmer sequence which is a mixed DNA and 2’-O-methyl RNA oligonucleotide,
and it avoids any unwanted targeting in the nucleus. However, the synthesis of such sequence is not
convenient, and the application in yeasts has not been tested before, not to mention such single-stranded
nucleotides are hard to be generated and replicated in vivo.

The limitations of direct introduction of PAMmer sequence into yeast create a niche for us to find
alternative solutions: can we instead generate ssDNA within yeasts, as an integrated part of their
metabolic activities? It turns out that generating ssDNA in yeast cells is viable. SCRIBE (Synthetic
Cellular Recorders Integrating Biological Events)^[8], intrinsically an in vivo reverse transcription
system, can reversely transcribe ssDNA with plasmids as the template^[9]. More importantly, the RNA
templates for reverse transcription are highly modularized, enabling effortless ssDNA switching and
thus the flexibility of mRNA targeting. In practice, the design of molecular cloning is relatively
convenient as well: a retron and an RNA template of the wanted ssDNA sequence can be assembled on the
same plasmid, enabling a one-step transformation in yeasts.

Coupling with cellular suicidal system

Mutations in the targeted gene will cause instant translocation of suvCas9 complex, switching on the
suicidal gene expression to kill the mutated cell. Here we design to fuse dCas9 with the GAL4 binding
domain and its activation domain on the foundation of yeast one-hybrid system^[13] that allows
the interaction of DNA-binding domain and the isolation of the targeted promoter and the downstream
gene. With the engineered Saccharomyces cerevisiae background strain^[13] for one-hybrid system, the
interaction between the DNA-binding domain and the promoter containing recognition site will drive the
transcription of Thymine Kinase (TK) and RFP. Thymine Kinase^[14] is a novel selection marker in yeast,
which is analogous to the widely used URA3 auxotrophy marker^[15], allowing both selection and
counter-selection in respective media. In the selection media, which is YP-glycerol with antifolates,
only yeast populations that produce thymine kinase can survive, while on the counter-selection media,
which is SC media with FUdR, only the absence of TK can ensure the viability of yeast. Mutations
in targeted genes will cause sequence changes in mRNA, which significantly decrease the binding
activity of suvCas9 onto mRNA. The previously sequestered suvCas9 complex will be transported into
nuclear immediately in the guidance of GAL4 binding domain and the exogenous Nuclear Localization
Sequence (NLS), and turn on the transcription of TK and RFP gene, which in turn cause the death of
cell on the counter-selection media and the fluorescence expression that can be quantitatively detected
by FACS for further optimization of our surveillance system.


Figure 1A. A schematic overview of the targeting mechanism of suvCas9. suvCas9 can bind to a correct
mRNA in complex with sgRNA and PAMmer (illustrated above), which can then be translocated into the
nucleus and initiate a suicidal program whenever the mRNA is mutated (not shown). Specially designed
PAMmers will not disrupt the genomic sequence.


Figure 1B. Mechanism of the generation of ssDNA in vivo. A single strand DNA can be derived
from the reversely transcribed product by the retron (RT). Figures are adapted from (7) and (8).

Reference

Results

In vivo experiment of dCas9 localization guided by sgRNA in S. cerevisiae

In order to achieve the surveillance function of the whole circuitry, it is of great significance to indicate
the precise spatial distribution of suvCas9. With suvCas9 fused with GFP, we can easily detect the cellular
localization of suvCas9 by fluorescent microscopy. We observed perfect nucleus localization of suvCas9
without the presence of sgRNA or PAMmer. However, after the co-transformation of actin transcript targeted
sgRNA expression plasmid and the electroporation of PAMmer, a certain population of yeast cells showed
obvious appearance of cytoplasmic suvCas9 complex, which implied that the specificity of suvCas9 is
determined by sgRNA as well as PAMmer. More importantly, suvCas9 protein, sgRNA and PAMmer form a
complex that can be trapped into the cytoplasm by sgRNA-targeted mRNA.

Yeast one-hybrid system for suicide gene activation (URA3)

To test if the GAL4 binding domain and its activation domain fused dCas9 can actually activate
downstream gene expression. The background yeast strain we used contains SPAL:URA3 engineered
into S. cerevisiae genome. Interaction between suvCas9 protein complex and SPAL promoter will drive
the expression of URA3, therefore ensure the viability on autotrophy selection plates. The results
indicated that successful expression of as well as the nucleus localization of suvCas9 protein complex,
would guarantee downstream gene activation based on yeast one-hybrid system, which is the premise of
suicidal gene activation once the potential mutation occurs and suvCas9 recover its nuclear targeting
capability.

Validation of TK lethality and viability in different culture media

Thymine Kinase (TK), as a novel selection marker in yeast, allows both selection and counter-selection
in respective media. We validated the lethality and viability in different culture media of TK expressing
strains. In YP-glycerol with antifolates, the selection media, yeast populations that produce TK can
survive, while on the counter-selection media, SC media with FUdR, TK would cause exclusive lethality
in those populations. Therefore, TK can act as a candidate downstream suicidal gene to be activated
in the mutated cells.

proof of concepts

suvCas9 relocalization

In the presence of suvCas9s alone, GFP signal (indicating suvCas9) can only be observed in the nucleus,
indicating an enrichment of nuclear localization of suvCas9s. However, when small guide RNA
targeting the Actin message (sgActin) is co-expressed with suvCas9, GFP signal is relocated into
the cytoplasm. In contrast, after introducing PAMmers, which function as single-stranded DNA mimics,
the majority of suvCas9 proteins can now be stably sequestered in the cytoplasm.


Figure 1. Relocation into the cytoplasm of suvCas9 proteins can be orchestrated by single guide
RNAs targeting the Actin message (sgActin) and PAMmers.


As negative controls, when suvCas9 is expressed alone, or co-expressed with small guide RNA targeting
anti-sense message Actin mRNA sequence (sgNC), the GFP signal (indicating suvCas9) are strictly sequestered
in the nucleus, suggesting without the companion of sgRNA targeting sense Actin message, suvCas9 will be
translocated into the nucleus by its engineered NLS (nucleus localization sequence). As a positive control,
when yeasts are only transformed with a GFP expression construct, without the guidance of the NLS, the GFP
signal is limited within the cytoplasmic region.


Figure 2. Negative control and positive control for the relocation assay.

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