Page 91 - ebook

The development of RNA direct editing via TYPE-VI CRISPR system

Seong-Ho Park1, Seokju Park4, Jin Wu Nam4,5,6, Junho K Hur1,3, *

E-mail: juhur@hanyang.ac.kr
1 Department of Medicine, Major in Medical Genetics, Graduate School, Hanyang University, Seoul, Korea.
2 Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul, Korea.
3 Department of Genetics, College of Medicine, Hanyang University, Seoul, Korea.
4 Department of Life Sciences, College of Natural Science, Hanyang University, Seoul 04736, Republic of Korea.
5 Research Institute for Natural Sciences, Hanyang University, Seoul 04736, Republic of Korea.
6 Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04736, Republic of Korea.

Abstract We designed guideRNA(gRNA) for analyzed cleavage site and off-target of CasRX. The gRNA was
designed 12 gRNA in B4GALNT1 exon2 region. As the CRISPR/Cas9 system was recognized PAM
sequence, CasRX of type-VI was recognized 1 nucleotide sequence that called PFS. It is known that CasRX
The CRISPR/Cas systems are known as the 3rd generation gene-editing tool, and have been mostly didn’t have a preferred PFS sequence, so three gRNAs were designed for each PFS sequence (figure 1. C).
applied for DNA editing. CRISPR/Cas systems are widely studied in biological and medical research fields
as the CRISPR systems can conduct direct editing of DNA to permanently change the genome. However, A B4GALNT1
one o the safety concerns of the CISPR/Cas systems have been off-target effects, that induces 2.0
unanticipated DNA sequence changes in the genome. Many studies have been trying to overcome the
off-target issue by developing highly precise CRISPR/Cas systems for gene therapy. To this end,
CRISPR-Cas13, an RNA editing CRISPR system, had been studied as a tool for targeted gene regulation 1.5
without DNA changes. However, the Cas13 had not widely used research because of low RNA editing
efficiency. According to a recent study, the CasRX, a type of Cas13d, was high efficiency and small size,
it was an advantage in virus loading. In this study, we performed that degradome-seq for detected of the Gene expression level (normalized to GAPDH) 1.0
cleavage site in RNA for observed the off-target effect, the disadvantage of the CRISPR/Cas system, in
CasRX. And we researched guide RNA screening for optimized gRNA design.
Introduction 0.5
Previous studies have identified various CRISPR/Cas systems that have been utilized in various research
fields. In some applications of CRISPR systems demonstrated targeted genome editing via DNA 0.0
double-strand cleavage at the target site. Such genome editing systems were further developed where M NC g1 g2 g3 g5 g6 g7 g9 g10 g11 g13 g14 g15
only one or a few nucleotide sequences could be replaced [4]. In other applications, CRISPR variants that PFS C PFS G PFS U PFS T
were catalytically inactive forms were applied to harness their target DNA binding abilities to tether CasRX - + + + + + + + + + + + + +
epigenetic effectors for modulating the expression of the target gene[2][3]. While CRISPR systems that gRNA - - + + + + + + + + + + + +
target the DNA provide versatile tools for genome editing, medical applications of the CRISPR systems B
are prone to the risks of altering the DNA sequence and epigenetic markers at unanticipated genomic loci.
As a route to reduce the danger of changing the DNA some CRISPR applications target RNA for targeted
gene regulation. Some recent studies showed that CasRX, a type-IV CRISPR system, could be applied
to inhibit the expression of target gene by cleavage of target mRNA, blocking the splicing, and inhibiting
the translation (figure 1. A)[1]. Unlike the CRISPR systems that directly edits DNA, medical application of
CasRX may cause less safety concerns as the off-target effects do not permanently change the DNA.
Nonetheless, the off-target effects of CasRX could still induce changes in expression levels of unanticipat-
ed genes and raise safety concerns for therapeutic application. Moreover, the off-target effects of CasRX
at transcriptome-level is fully understood yet. To address the issue, we sought to conduct analyses of the
off-target effect of CasRX via Degradome-seq[5]. In this study, we applied high throughput sequencing
analyses for profiling the CasRX mediated cleavage positions of the on- and off-target mRNAs. (figure 1.
B). We anticipate that result will provide insight for designing CasRX guide RNAs with high efficiency and C
low off-target effects.
Results
A B CasRx cleavage site mRNA
CasRX
gRNA 5’ Cap CasRX AAAA(A)n
5’ 3’ 5’ PO4
5’ Cap AAAA(A)n
HPEN ssRNA
cleavage RNA adaptor 5’ PO4
site 5’ 3’ OH AAAA(A)n
RNA ligation Reverse transcription
5’ AAAA(A)n
3’ TTTT Figure 2.
pre-mRNA Second cDNA synthesis oligo dT adaptor primer We transfected CasRX plasmid and gRNA plasmid into HEK 293T cell for measured knock-down efficiency
gRNA dCasRX 5’
3’ of each gRNA. After 2days of transfection, We harvested cell and RNA isolation. We performed qRT-PCR
MmeI digestion Wafter cDNA synthesis. Compared to mock(M), which is a cell treated with reagent only, the knock-down
20bp
MmeI efficiency was shown in the sample treated with gRNA and CasRX, and the kD efficiency was about 70% for
5’ 3’
Splicing 3’ 5’ all gRNAs without PFS sequence specificity (figure 2. A).
3’ dsDNA adaptor ligation According to qRT-PCR, We performed degradome-seq. The mRNA was isolated from 30 ug of initial input
5’ 3’ RNA using poly(T) beads. The 5' RNA adapter was ligated to the cleaved mRNA without the 5' cap. After
mature mRNA 3’ 5’ 3’ dsDNA adaptor cDNA synthesis using poly(T) adaptor primer, We restricted cDNA with a sticky end about 20 bp away from
5’ 3’ the 5' end of the cleavaged mRNA using the MmeI recognition site of the 5' RNA adapter. After ligated 3’
3’ 5’
Ribosome gRNA dCasRX PCR dsDNA adaptor, Electrophoresis using 12% TBE-PAGE gel, and gel extraction between 50 and 75 bp
5’ 3’ index because the product size is 63 bp (figure 2. B). Using the extracted DNA, an index capable of recognizing
3’ 5’ the sequencing library was attached using PCR, 6% TBE-PAGE gel electrophoresis was performed, and the
Translation library was gel extracted(Figure 2. C).
bloking Sample pooling and sequencing We performed deep sequencing of library using iseq 100(illumina). We have analyzed deep sequencing
C B4GALNT1 data, and based on this data, we would like to present a high-efficiency gRNA design guideline.
References
EXON 2
g1 g3 g2
g5 g7 g6 [1] Silvana Konermann, Peter Lotfy, Nicholas J. Brideau, Jennifer Oki, Maxim N. Shokhirev, Patrick D. Hsu, Transcriptome
g9 g10 g11
g14 g13
g15 Engineering with RNA-Targeting Type VI-D CRISPR Effectors, Cell, Volume 173, Issue 3, 2018, Pages 665-676.e14,
[2] Jaehoon Choi, Taegeun Bae, Ninj Byambasuren, Seong-Ho Park, Chris H. Jo, Dokyoung Kim, Junho K. Hur, Nathaniel S.
Hwang, CRISPR-Cpf1 Activation of Endogenous BMP4 Gene for Osteogenic Differentiation of Umbilical-Cord-Derived
Mesenchymal Stem Cells, Molecular Therapy - Methods & Clinical Development, Volume 17, 2020, Pages 309-316,
[3] Thakore, P., D'Ippolito, A., Song, L. et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of
distal regulatory elements. Nat Methods 12, 1143–1149 (2015).
[4] Doudna, J.A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).
[5] Chanseok Shin, Jin-Wu Nam, Kyle Kai-How Farh, H. Rosaria Chiang, Alena Shkumatava, David P. Bartel, Expanding the
Figure 1. MicroRNA Targeting Code: Functional Sites with Centered Pairing, Molecular Cell, Volume 38, Issue 6, 2010, Pages 789-802,