Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

Abstract

The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 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 offtarget cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 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-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less offtarget mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable 2 diseases caused by genetic mutation.

Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

“…The importance of RNA A-form helical structure and 2′-OH chemistry has been partially investigated for pseudoknot stability and protein interaction 85,86 , but not in the context of CRISPR-Cas systems. The unique noncanonical nature of the Cas12a 5′ pseudoknot, as well as previous attempts at modification 74,78,81 , suggested that it would be challenging to modify for therapeutic development. To focus on the pseudoknot structure and uncouple it from guide region effects, we maintained an entirely native RNA ribose (2′-OH) guide region throughout most of this study.…”

“…In this study, we have used chemical modification to investigate the necessity of the ribose 2′-OH group in the AsCas12a crRNA 5′ pseudoknot structure for enzyme activity. The primary question was whether the pseudoknot structure was too complex or sensitive for efficient chemical modification, which previous studies had suggested 74,78,81 . The rationale for our approach is the high likelihood that therapeutic applications of Cas12a that require direct delivery of the crRNA will also require heavy or complete chemical modification, as is the case for siRNAs.…”

“…More complete or systematic modification of Cas9 and Cas12a guide RNAs has been explored using a variety of chemistries. DNA (2′-deoxy) has been reported to alter enzyme activity or specificity while helping to probe 2′-hydroxyl requirements of the enzyme 69,[78][79][80] . A broader array of modifications, like 2′-F, 2′-Omethyl, bridged nucleic acid (BNA), UNA, LNA, 2′F-arabinonucleic acid (FANA), and PS have been tested alone or in combination as well 61,70,71,[81][82][83][84] .…”

“…These studies largely demonstrated that much of the guide could be modified, with moderate to high editing preserved, but particular positions, such as seed regions or individual residues, required RNA nucleotides. Specificity could also be improved with careful placement of modifications in the guide region 78,79,83,84 . For Cas9, retention of editing activity has been suggested to correlate with conserved polar contacts between the protein and the hydroxyl at the ribose 2′ position and the need to retain A-form-like helical architecture [69][70][71]80 .…”

“…For Cas12a, attempts to fully modify the crRNA guide have proven unsuccessful 74,78,81 . However, the 5′ pseudoknot handle posed the greatest challenge as it appeared to tolerate very little modification to the ribose, suggesting that successful modification of this unique structure would be the major limitation to greater modification of Cas12a guides for therapeutic development.…”