Page 26 - L. Genetics and genomics

[L. Genetics and genomics-21]

Enhancement of target specificity of CRISPR–Cas12a by using

a chimeric DNA–RNA guide

Hanseop Kim¹˙², Wi-jae Lee¹˙³, Yeounsun Oh¹˙⁴, Seung-Hun Kang¹˙⁵, Kyung-Seob Lim¹, Young-Ho Park¹,
Bong-Seok Song¹, Yeung Bae Jin⁶, Dong-Seok Lee², Sun-Uk Kim¹˙⁷, Seung Hwan Lee⁶

¹Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology
(KRIBB), Cheongju 28116, Republic of Korea, ²School of Life Sciences and Biotechnology, BK21 Plus KNU Creative
BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea, ³Department of Bioscience

and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea, ⁴Department of Biotechnology, College of
Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea, ⁵Department of Medicine,
Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea, ⁶National Primate Research Center
(NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Republic of Korea, ⁷
Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology
(UST), Daejeon 34113, Republic of Korea

The CRISPR–Cas9 system is widely used for targetspecific genome engineering. CRISPR–Cas12a (Cpf1) is one of the

CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM)
sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target

sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM
sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when

Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and
modified crRNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations

in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA
to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided

CRISPR–Cas12 and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based
CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential

for therapeutic applications in diseases caused by genetic mutations.