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. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a 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 off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a 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–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

Chimeric crRNA improves CRISPR–Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2

“…We have designed and compared three different types of N501Y crRNA, namely, N501Y crRNA 20-nt (20-nt spacer, designing 20-nt spacer crRNAs is the traditional strategy for genome editing and detection by CRISPR-Cas12a, and in accordance, this 20-nt crRNA has recently been used to detect N501Y in miSHERLOCK platform [ 7 ]), N501Y chimeric crRNA 24-nt (24-nt spacer), and N501Y crRNA 24-nt (24-nt spacer) to determine the most efficient crRNA used for N501Y detection (The sequences of primers and crRNAs are given in Table 1 ). N501Y chimeric crRNA 24-nt was designed according to the method by Kim et al replacing the last 8-nt of the crRNA with DNA which can improve CRISPR-Cas12a specificity of target DNA cleavage [ 9 ]. However, whether this kind of chimeric crRNA can increase the specificity of detection (collateral effect of Cas12a) has not been determined as per our knowledge.…”

“…The sensitivity of chimeric crRNA is comparable to regular crRNA, whereas the specificity is higher and can be stable for more than 2 h. It improves and provides the versatility of applying CRISPR-Cas12a in a larger throughput, even as a point-of-care testing (POCT). Replacing the last 8-nt of the crRNA with DNA can decrease the binding energy between crRNA and target DNA, leading to less off-target of CRISPR-Cas12a [ 9 ]. RNA-guided Cas12a unleashes indiscriminate single-stranded DNase activity when CRISPR-Cas12a recognizes its target [ 11 ].…”

“…It has been applied to SARS-CoV-2 and/or variants detection, including Cas9 (RAY [ 5 ]), Cas12a (DETECTR [ 6 ] and miSHELOCK [ 7 ]), and Cas13a (SHERLOCK [ 8 ]). Among them, the Cas12a system is faster than Cas13a as Cas13a requires in vitro transcription for detection, and possess higher target specificity than CRISPR-Cas9 system as the latter is more tolerant to mismatch [ 9 ]. Thus, CRISPR-Cas12a was used in our study.…”

Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay

“…Also, the AIOD-CRISPR assay showed a high specificity in the SARS-CoV-2 detection without any cross-interaction with other sequences (e.g., SARS-CoV, MERS-CoV; Fig. 3a), which may be due to the nature of the CRIPSR-Cas12a's singlebase specificity 28,29 .…”

CRISPR-based strategies for targeted transgene knock-in and gene correction

“…Immunogenicity issue can also be circumvented by transient modulation of the genes involved in immunity by using a CRISPR-based synthetic repressor 191 . Other strategies to minimize undesired off-target mutagenesis are using light-activated, chemical-inducible, or multiple-input logic gate genetic circuits to spatiotemporal and conditional control of CRISPR/Cas9 activities 192 ; cell type- or tissue-specific promoters and delivery vehicles (for example, specific viral serotypes 16 , 17 , 193 or ligand-targeted cargos 192 ), engineered high-fidelity Cas9 variants 194 – 200 , and rational chemical modifications to the sgRNA 192 ; a chimeric DNA-RNA guide 201 ; and engineered truncated sgRNA 202 , 203 and sgRNA secondary structures 204 . Onsite mutagenesis and heterogeneity can also occur in the uncorrected population of cells following CRISPR/Cas9-induced double-stranded DNA breaks 99 .…”