Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease

• DOI: 10.1016/j.celrep.2022.111418
• Publication Date: 2022-09-27T14:36:17Z
• Authors: Yujue Wang; Yannan Wang; Deng Pan; Haopeng Yu; Yifei Zhang; Weizhong Chen; Fan Li; Zhaowei Wu; Quanjiang Ji
• Keywords: Trans-activating crRNA; Genome Engineering; Guide RNA; Nuclease

Gene therapy is limited by inefficient delivery of large clustered regularly interspaced short palindromic repeat (CRISPR) effectors, such as Cas9 and Cas12a nucleases. Cas12f nucleases are currently one the most compact CRISPR genome editors. However, available toolkit efficient editors limited. Here, we report characterization engineering a miniature CRISPR-Cas12f system from Syntrophomonas palmitatica (SpaCas12f1, 497 amino acids). We show that CRISPR-SpaCas12f1 cleaves double-stranded DNA (dsDNA) with 5′ T-rich PAM specificity naturally active for editing in bacteria. identify trans-activating RNA (tracrRNA) harbors unique head-to-toe hairpin structure, natural structure key factor restricting SpaCas12f1 human cells. Systematical guide transforms into an editor comparable to Francisella novicida CRISPR-Cas12a. Our findings expand mini toolbox, paving way therapeutic applications manipulation technologies.