Past, present, and future of CRISPR genome editing technologies

Cell, Год журнала: 2024, Номер 187(5), С. 1076 - 1100

Опубликована: Фев. 1, 2024

Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes treat underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency easy programmability, stand at forefront this revolution. In Review, we discuss current state CRISPR gene technologies both research therapy, highlighting limitations that constrain them technological innovations have developed recent years address them. Additionally, examine summarize landscape applications context health therapeutics. Finally, outline potential future developments could shape coming years.

Язык: Английский

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RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2

Nature, Год журнала: 2023, Номер 613(7944), С. 582 - 587

Опубликована: Янв. 4, 2023

Abstract Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, DNA and double-stranded following recognition complementary RNA target, culminating in abortive infection 1 . Here we report structures binary, ternary quaternary complexes to reveal complete activation pathway. Our autoinhibited until binding cognate which exposes the RuvC active site within large, positively charged cleft. Double-stranded substrates are captured through duplex distortion local melting, stabilized by pairs ‘aromatic clamp’ residues crucial for vivo immune system function. work provides structural basis this mechanism achieve population-level immunity, can be leveraged create rational mutants degrade spectrum collateral substrates.

Язык: Английский

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Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(20)

Опубликована: Март 22, 2024

Abstract The CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐ cleavage activity of AsCas12a and LbCas12a discover that they can directly activated by full‐size RNA targets, although exhibits weaker than both single‐stranded substrates. Remarkably, find RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared activation. Based these findings, develop “ U niversal N uclease I dentification V irus E mpowered R NA‐ Se nsing” (UNIVERSE) assay acid testing. We incorporate T7 transcription step into this assay, thereby eliminating requirement protospacer adjacent motif (PAM) sequence target. Additionally, successfully detect multiple PAM‐less targets HIV clinical samples are undetectable conventional based double‐stranded activation, demonstrating unrestricted selection with UNIVERSE assay. further validate utility testing HPV 16 samples. envision targeting capability may bring paradigm shift Cas12a‐based detection enhance understanding CRISPR‐Cas biochemistry.

Язык: Английский

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CRISPR/Cas12a-derived ratiometric fluorescence sensor for high-sensitive Pb2+ detection based on CDs@ZIF-8 and DNAzyme

Biosensors and Bioelectronics, Год журнала: 2024, Номер 251, С. 116089 - 116089

Опубликована: Фев. 3, 2024

Язык: Английский

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Trans-nuclease activity of Cas9 activated by DNA or RNA target binding

Nature Biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Май 29, 2024

Язык: Английский

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Structure-guided discovery of anti-CRISPR and anti-phage defense proteins

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Янв. 20, 2024

Язык: Английский

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CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics

ACS Synthetic Biology, Год журнала: 2024, Номер 13(3), С. 837 - 850

Опубликована: Фев. 13, 2024

The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different of cancers, there is a pressing need an easy-to-use, multiplex point-of-care diagnostic platform detecting types. Recently, CRISPR-Cas systems have been repurposed detection. Here, we established multiplexed assay (CRISPRD) to detect cervical cancer-causing hrHPVs one reaction (one-pot assay). We harnessed the compatibility thermostable AapCas12b, TccCas13a, HheCas13a nucleases with isothermal amplification successfully detected HPV16 HPV18, along internal control single-pot limit detection 10 copies 100% specificity. This offers rapid practical solution hrHPVs, which may facilitate large-scale screening. Furthermore, CRISPRD programmability enables it be adapted any two nucleic acid biomarkers as well control.

Язык: Английский

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DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity

Nature Microbiology, Год журнала: 2024, Номер 9(5), С. 1368 - 1381

Опубликована: Апрель 15, 2024

Язык: Английский

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The Role of Biomarkers in HPV-Positive Head and Neck Squamous Cell Carcinoma: Towards Precision Medicine

Diagnostics, Год журнала: 2024, Номер 14(13), С. 1448 - 1448

Опубликована: Июль 7, 2024

Head and neck cancer (HNC) represents a significant global health challenge, with squamous cell carcinomas (SCCs) accounting for approximately 90% of all HNC cases. These malignancies, collectively referred to as head carcinoma (HNSCC), originate from the mucosal epithelium lining larynx, pharynx, oral cavity. The primary risk factors associated HNSCC in economically disadvantaged nations have been chronic alcohol consumption tobacco use. However, more affluent countries, landscape has shifted identification human papillomavirus (HPV) infection, particularly HPV-16, major factor, especially among nonsmokers. Understanding evolving distinct biological behaviors HPV-positive HPV-negative is critical developing targeted treatment strategies improving patient outcomes this complex diverse group cancers. Accurate diagnosis essential comprehensive model that integrates molecular characteristics, immune microenvironment, clinical outcomes. aim review was summarize current knowledge advances DNA, RNA, protein biomarkers bodily fluids tissues introduced new possibilities minimally or non-invasive diagnosis, monitoring, assessment therapeutic responses.

Язык: Английский

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TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Июль 13, 2024

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram from diverse Cas12 nucleases, an dsDNA and subsequent collateral single-stranded DNA cleavage-all without RNA necessarily encoding protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, demonstrate PAM-independent detection with Cas12b, Cas12e, Cas12f nucleases. Furthermore, rationally truncating target boosts activity, while absence gRNA reduces background activity enhances sensitivity. Finally, apply this platform detect 16 S rRNA sequences five different bacterial pathogens universal tracrRNA. These findings extend tracrRNA reprogramming dsDNA-targeting expanding flexibility versatility CRISPR-based detection.

Язык: Английский

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