Direct Observation of DNA Target Searching and Cleavage by CRISPR-Cas12a

Jeon, Young Hoon; Choi, You Hee; Jang, Yunsu; Gu, Jiyoung; Jeong, Cherlhyun; Lee, Sang-Hwa; Bae, Sangsu

doi:10.1016/j.bpj.2018.11.1166

Cited by 22 publications

(62 citation statements)

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“…This model ( Fig. 1 and SI Appendix) is based on recent structural biology and single-molecule studies (38,39) which revealed that DNA hydrolysis by Cas12a occurs in three discrete stages: "PAM attachment," where Cas12a latches onto a PAM site; "crRNA-DNA inspection," where Cas12a forms a partial crRNA-DNA hybrid; and "reconfiguration," where the protein forms a ternary complex and undergoes a conformal change that exposes its catalytic residues. While the final DNA cleaving step occurs after approximately 1 min under the conditions tested in ref.…”

Section: Resultsmentioning

confidence: 99%

“…While the final DNA cleaving step occurs after approximately 1 min under the conditions tested in ref. 38, Cas12a molecules with inactivated nuclease sites remain stably bound to their DNA target for more than 500 s. Hence, the reconfiguration step effectively has no detectable off rate, suggesting that DNA cleavage may be inevitable (given enough time) after Cas12a has reached this stably bound ternary state. The same stability has also been observed in single-molecule Cas9 experiments (37).…”

Section: Resultsmentioning

confidence: 99%

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Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

Specht

Lambert

2020

Proc. Natl. Acad. Sci. U.S.A.

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The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) fromFrancisella novicidaas it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA–DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

Specht

Lambert

2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The process by which the CRISPR effectors find their target has also received a lot of (35,37,87,88). For both, there is a first step of 3D-diffusion until a DNA molecule is encountered (35).…”

Section: Switching Crispr On and Off: A Matter Of Timingmentioning

confidence: 99%

CRISPR Tools To Control Gene Expression in Bacteria

Vigouroux

Bikard

2020

Microbiol Mol Biol Rev

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SUMMARY CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.

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“…On the other hand, the ampli ed products generated during the RPA continuously trigger CRISPR-Cas12abased collateral cleavage activity. Previous studies 14,17 have demonstrated that the collateral cleavage activity of the CRISPR-Cas12a system is independent of target strand cleavage. Therefore, target sequences for our AIOD-CRISPR assay are not limited by the Cas12a's protospacer adjacent motif (PAM) 22 .…”

Section: Resultsmentioning

confidence: 99%

“…and Cas13a, perform strong collateral cleavage activities in which the Cas nucleases activated by crRNAtarget duplex can indiscriminately cleave surrounding non-target single-stranded nucleic acids. 13,14,15,16,17 By combining with RPA pre-ampli cation, Cas13 and Cas12a nucleases have, respectively, been used to develop SHERLOCK (Speci c High-sensitivity Enzymatic Reporter UnLOCKing) system 18 and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) system 14 for highly sensitive and speci c nucleic acid detection. Apart from the RPA pre-ampli cation method, some CRISPR-Cas-based nucleic acid detection utilized LAMP and PCR pre-ampli cation, such as CRISPR-Cas12b-assisted HOLMESv2 platform and SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR).…”

Section: Introductionmentioning

confidence: 99%

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus at the Point of Care

Ding

Yin

et al. 2020

Preprint

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The recent outbreak of novel Coronavirus (SARS-CoV-2), the causative agent of COVID-19 disease, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and prevent disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for simple, rapid, ultrasensitive, specific, one-pot, and visual detection of coronavirus SARS-CoV- 2 and HIV-1 virus. In our AIOD-CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a-based detection and improve both detection sensitivity and fluorescence signals. The AIOD-CRISPR assay method was utilized to detect nucleic acids (DNA and RNA) of the SARS-CoV-2 and HIV-1 with a sensitivity of few copies. We validated our AIOD-CRISPR method by using COVID-19 swab samples and obtained consistent results with that of RT-PCR method. More importantly, we successfully demonstrated to use a low- cost hand warmer (~$ 0.3) as an incubator of our AIOD-CRISPR assay and detect COVID-19 patient samples within 20 minutes, enabling an instrument-free, visual detection of COVID-19 at the point of care. Thus, our method has significant potential for developing next-generation point-of-care molecular diagnostics.

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Direct Observation of DNA Target Searching and Cleavage by CRISPR-Cas12a

Abstract: Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNAguided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has

Cited by 22 publications

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Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

CRISPR Tools To Control Gene Expression in Bacteria

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus at the Point of Care

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