Engineering CRISPR/Cpf1 to maximize genome editing efficiency Bin Li1, Weiyu Zhao1, Xiao Luo1, Xinfu Zhang1, Chenglong Li2, Chunxi Zeng1, & Yizhou Dong1,3* 1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA. 2Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA. 3Department of Biomedical Engineering, The Ohio State University, Columbus, OH 43210, USA. CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR- associated proteins) system is part of adaptive immunity in bacteria and archaea. In 2015, two Cpf1 (CRISPR from Prevotella and Francisella 1) proteins from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) were discovered and displayed comparable genome editing capability to Cas9. Cpf1 is functional through a single CRISPR RNA (crRNA, Figure 1a), which consists of a 5’-handle (20 nucleotides) and a guide segment (23 nucleotides). To increase genome editing efficiency of CRISPR-Cpf1 system, we applied two engineering methods including chemical modifications and structural alterations. We systematically investigated 42 engineered crRNAs and established their structure-activity relationships. Subsequently, we identified ψ-modification was a favorable chemical modification for AsCpf1 mRNA. More importantly, combination of lead crRNA and ψ-mRNA significantly increased gene cutting efficiency over 300% compared to the control group. This combination induced more dramatic improvement of gene cutting efficiency when using LbCpf1 (Figure 1b). In addition, our results indicated that the applicability of crRNAs from Cpf1 orthologs is distinctly different between AsCpf1 and LbCpf1. LbCpf1 is more conservative for recognition of the loop structure at the 5’- handle. Consequently, our findings offer a promising strategy for broad genome editing applications. Figure 1. a. Schematic illustration of AsCpf1-crRNA-target DNA complex. b. Gene editing efficiency through combination of chemically modified crRNA and LbCpf1 mRNA for the human DNMT1-3 in HEK293T, Hep3B and U87 cells. .