Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Abstract

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.

A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration

“…The velocity of TSDR is significantly reduced when single-base mismatches occur on or near the toehold ( 25 , 38 ) To investigate whether TOA has a similar specificity, we compared the effects of four single-base mutations at different positions (Mutation-1, Mutation-2, Mutation-3 and Mutation-4) on the signal generated by TOA ( Supplementary Figure S3 ) and calculated the DFs (Figure 2 ).…”

“…The BM-TOA system possesses excellent single-base mismatch specificity without introducing any additional mismatches in crRNA ( 21 , 32 , 37 , 38 ) and can be widely used as a high-performance CRISPR–Cas12a platform for detection of various types of mutations. In addition, the system also proved the compatibility of the TOA activation mode: the TOA has the potential to cascade with various reaction networks.…”

“…In most post-PCR assays, ss-targets with hybridization activity are amplified to trigger the subsequent detection step, but it needs to unravel the potential self-hybridization of the targets by raising the reaction temperature ( 23 , 38 ). Fortunately, the main structure of the BMP-like target is a stable double strand almost without a troublesome secondary structure.…”

G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation

“…As an alternative approach, modifications of sgRNA are particularly attractive because of their simplified procedures and high gene delivery efficiency ( 17–21 ). Structure-based transformation of gRNA is an interesting theme and uses, for example, the hairpin on the 5′ end of gRNA ( 22 ) or antispacer peptide nucleic acids designed to improve the sequence recognition specificity ( 23 ). In addition, G-quadruplex-based motifs designed with gRNAs endowed higher stability against cellular nucleases ( 24 ), and engineered G-quadruplex structures in the 3' end of sgRNA or tracrRNA with chemical sensitivity were shown to modulate CRISPR-based genomic regulation ( 25 ).…”

Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering

“…Instead, we focus on strategies and implementations reported to increase editing specificity. The strategies utilized for reducing off-target effects include Cas9 nuclease engineering [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ], using natural Cas9 nucleases with high specificity [ 34 , 35 ], the utility of base editors [ 36 , 37 , 38 ] and prime editors [ 39 , 40 ], gRNA design optimization and/or modulation [ 8 , 9 , 41 , 42 , 43 , 44 , 45 , 46 ], control of Cas9′s activity via direct delivery (e.g., ribonucleoprotein (RNP) [ 20 , 47 ], virus-like particles [ 48 , 49 , 50 , 51 ], cell-penetrating peptide-mediated delivery [ 52 ], mRNA [ 53 ], and self-limiting circuits [ 54 ]), and combination with anti-CRISPR proteins or CRISPR inhibitors [ 55 , 56 , 57 , 58 ]. Among these strategies, direct or indirect engineering of Cas9 proteins represents the majority of efforts, evidenced by the relatively large number of studies toward this direction ( Table 1 ).…”