Page 25 - L. Genetics and genomics

Improvement of target specificity of CRISPR-Cas12a
by using chimeric DNA-RNA guide.
1,3
Hanseop Kim , Wi-jae Lee , Yeounsun Oh , Seung-Hun Kang ,Dong-Seok Lee , Sun-Uk Kim , Seung Hwan Lee 2
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1 Futuristic Animal Resource & Research Center (FARRC), Korea Researh Institute of Bioscience and Biotechnology(KRIBB), Cheongju, Korea
2 National Primate Research Center (NPRC), Korea Researh Institute of Bioscience and Biotechnology(KRIBB),Cheongju, Korea
3 Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon, Korea
4 Department of Medicine, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
5 School of Life Sciences and Biotechnology, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea
6 Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea
7 Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
B a c k g r o un d M e t h o ds
The CRISPR–Cas system is a bacterial immune system, which and it is now
widely used for target-specific genome editing in various organisms. One of
the CRISPR systems, CRISPR–Cas9 RNA-guided endonuclease, is routinely
used to specifically correct or control genes of interest based on its ability to
cut double-stranded DNA. Another component of the CRISPR system,
CRISPR–Cas12a (Cpf1)was recently reported and is a class II, typeVeffector
nuclease that has a bi-lobed structure composed of nuclease and recognition
domains, similar to Cas9. Cas12a binds the target DNA helix via a single-
stranded (cr)RNA, forming a DNA–RNA hybrid duplex. Cas12a has attracted
attention as an excellent genome-engineering tool as it overcomes certain
limitations of Cas9. In contrast to Cas9, which recognizes guanine (G)-rich
sequences, Cas12a recognizes specific thymine (T)-rich protospacer adjacent
motif (PAM) sequences (TTTN or TTN) and can specifically induce double-
strand DNA cleavage.
A im
The CRISPR–Cas12a is now broadly applicable for gene editing in various Partial DNA replacement introduced to the guide RNA of CRISPR-Cas12a.
organisms ranging from microorganisms to humans, and therefore, we In vitro cleavage and in vivo genome editing experiments were performed
investigated off-target cleavage by Cas12a and engineered the Cas12a with these chimeric DNA-RNA guides.
(cr)RNA to address the off-target cleavage issue.
R es ul t s

Figure 1. Time-course analysis of the on/off-target cleavage of Cas12a using chimeric DNA–RNA guides on various target sequences.
Target and offtarget DNA cleavage experiments to confirm the target specificity of AsCpf1 using a chimeric DNA–RNA guide (DNA substitutions in the 3’-end) on (A) DNMT1, (B) GRIN2B,
and (C) HPRT1 genes.

Figure 2. Cas12a editing efficiency and
specificity are enhanced by combination of a
chimericDNA–RNA guide and dead(d) /
nickase(n) form of SpCas9.
(A) Endogenous CCR5 gene editing in HEK293FT cell
using chimeric DNA–RNA guided Cas12a and d/n-SpCas9
combination. (B) Top: Efficiency of genome editing for on-
and off-target in CCR5 gene. Bottom: The off-target indel
frequency (%) was magnified. (C) Fold increase in the indel
ratio (%) between only Cas12a treated, Cas12a and dCas9
co-treated, and Cas12a and nCas9 co-treated samples. (D)
Determination of target specificity (on/off1 cleavage ratio)
calculated from (B).
C o n c l us io n
R ef er en c es A c k n ow l edg emen t s

Authors thank Dr Kyu-Tae Chang and other
Zetsche,B., Gootenberg,Jonathan S.,
Abudayyeh,Omar O., Slaymaker,IanM., members of the National Primate Research Center
Makarova,Kira S., Essletzbichler,P., (NPRC) for helpful discussions.
Volz,Sara E., Joung,J., van der Oost,J.,
Regev,A. et al. (2015) Cpf1 is a single
RNA-guided endonuclease of a class 2
CRISPR-Cas system. Cell, 163, 759– C o n t a c t In f or ma t ion
771.
Yin,H., Song,C.-Q., Suresh,S., Kwan,S.-
Y., Wu,Q., Walsh,S., Ding,J., To whom correspondence should be addressed.
Bogorad,R.L., Zhu,L.J., Wolfe,S.A. et al.
(2018) Partial DNA-guided Cas9 enables Seung Hwan Lee. Tel: +82 43 240 6307; Fax: +82
genome editing with reduced off-target 43-240 6309; Email: lsh080390@kribb.re.kr
activity. Nat. Chem. Biol., 14, 311
Dong-Seok Lee. Tel: +82 53 950 7366; Fax: +82 53
Chen,F., Ding,X., Feng,Y., Seebeck,T.,
Jiang,Y. and Davis,G.D. (2017) Targeted 943 6925; Email: lee1@knu.ac.kr
activation of diverse CRISPR-Cas
Mechanism underlying the enhanced Cas12a editing specificity systems for mammalian genome editing Sun-Uk Kim. Tel: +82 43 240 6321; Fax: +82 10
9419 7993; Email: sunuk@kribb.re.kr
by using chimeric DNA–RNA guide. via proximal CRISPR targeting. Nat.
Commun., 8, 14958.
In this model, when the 3'-end DNA substituted crRNA was applied to the Cas12a
system, the principle that the off-target mutation was reduced compared to wild-
type crRNA was explained by the change of the hybridization energy between
crRNA and target DNA strand.