Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a

Abstract

Genome editing has recently made a revolutionary development with the introduction of the CRISPR–Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3′ end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

“…In other words, that it is the synSeparator downstream of the GFP-targeting spacer, rather than the one upstream, that improves performance by remaining attached to the post-processed GFP-targeting spacer. This would be in line with studies showing that crRNA performance can be improved by RNA 3' spacer extensions (Creutzburg et al, 2020;Kocak et al, 2019;Nguyen et al, 2020). However, our results demonstrate that the synSeparator exerts its beneficial effect before or during crRNA processing, rather than by modifying the 3' end of the post-processed GFP-targeting spacer itself ( Fig.…”

“…1K). Furthermore, previous data show that Cas12a processing is sensitive to RNA secondary structure (Creutzburg et al, 2020).…”

MeCas12a, a Highly Sensitive and Specific System for COVID‐19 Detection

“…The CRISPR/Cas12a‐based detection readout signal relies on the crRNA‐guided targeting cleavage efficiency, which is affected by the spacer sequence and secondary structure of crRNA. [ 21 ] We then tested the abilities of various crRNAs to guide Cas12 to detect synthetic ssDNA fragments of their respective E genes, and found SC2‐crRNA2–4‐ and MC‐crRNA1–3‐induced stronger fluorescence (Figure S2a,b, Supporting Information), which were chosen for subsequent experiments. The potential off‐target was analyzed, and no cross‐reaction was detected (Figures S1d,e and S2c,d, Supporting Information).…”

Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER

“…S2 and S3 online). As the CRISPR/Cas12a-NER readout signal relies on the crRNA-dependent targeting cleavage efficiency, which is affected by the secondary structure and spacer sequence of the crRNA [7,8]. The crRNAmix (equal mix work crRNAs targeting at each gene, named orf1a-crRNAmix, orf1b-crRNAmix, N-crRNAmix, E-crRNAmix) mediated the strongest fluorescence signal (Fig.…”