Qixiao Lin

Southern Medical University

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Zhili Rong

Southern Medical University

Guanjie Huang

Southern Medical University

Jiawei Liu

Southern Medical University

Shufeng Ma

Southern Medical University

Xiaoyang Zhao

Henan Polytechnic University

Zhuo Ma

Nanjing University of Posts and Telecommunications

Yuchen Liu

University of California, Davis

Mengrao Li

Southern Medical University

Tao Huang

Wenzhou Medical University

Zhihong Tan

Southern Medical University

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Southern Medical University

Guangzhou Medical University

Guangzhou Regenerative Medicine and Health Guangdong Laboratory

Southern Medical University Shenzhen Hospital

Kunming University of Science and Technology

Dongguan People’s Hospital

Capital Medical University

University of Electronic Science and Technology of China

Nanfang Hospital

Nanjing Medical University

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Additional file 1 of Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing

Figshare (2023)

Xin Zhang Xinlong Wang Jie Lv Hongxin Huang Jiahong Wang

Additional file 1: Fig. S1 Establishment of the dLbCas12a-p300knock-in HEK293T cell line. Fig. S2 Circular gRNAs increase thetranscription efficiency of LbCas12a-based activators. Fig. S3 Establishment of the LbCas12a knock-in HEK293Tcell line and off-target analysis of LbCas12a with different gRNAs. Fig. S4 Circular gRNAs improve the DNA cleavage efficiency of LbCas12ain MCF7 cells. Fig. S5 comparison between cgRNA with other extendedstructuralized gRNAs. Fig. S6 Circular gRNAs improve the gene expressionor DNA cleavage of AsCas12a-based effectors. Fig. S7 Multiplexedgene activation and cleavage guided by cgRNAs. Fig. S8 Efficient andspecific RNA cleavage activity of CasRX with cgRNA. Fig. S9 Thetrans-cleavage activity of CasRx–mediated exogenous transcripts degradation. Fig.S10 The trans-cleavage activity of CasRx–mediated endogenous transcriptsdegradation. Fig. S11 The structures of circular gRNAs for LbCas12awith different linkers targeting IL1RN predicted by mFold. Fig. S12The structures of circular gRNAs for LbCas12a with different linkerstargeting IL1RN predicted by mFold. Fig. S13 The structures ofcircular gRNAs for CasRx with different linkers targeting STAT3predicted by mFold.

Guide RNA

10.6084/m9.figshare.23573594

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Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing

Genome biology (2023)

Xin Zhang Xinlong Wang Jie Lv Hongxin Huang Jiahong Wang

Abstract Background The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability. Results Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA. Conclusions CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.

Guide RNA

CRISPR interference

Trans-activating crRNA

Nuclease

10.1186/s13059-023-02992-z

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Additional file 2 of Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing

Figshare (2023)

Xin Zhang Xinlong Wang Jie Lv Hongxin Huang Jiahong Wang

Additional file 2: Table S1 The sequences of sgRNAs, primers and linkers.

Guide RNA

CRISPR interference

10.6084/m9.figshare.23573597

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