* . *
  • About
  • Advertise
  • Privacy & Policy
  • Contact
Saturday, June 21, 2025
Earth-News
  • Home
  • Business
  • Entertainment
    ‘Puttin’ on the Ritz’: Civic Players bring ‘Young Frankenstein’ to life – Yahoo

    Civic Players Deliver a Hilarious and Unforgettable Performance of ‘Young Frankenstein

    ‘Wheel of Fortune’: Amputee Wins $60,000 After Breaking Incredible ‘Curse’ – Hastings Tribune

    Wheel of Fortune’ Amputee Breaks Incredible ‘Curse’ to Win $60,000!

    North Star Sports & Entertainment Network: Coming soon – KTTC News

    North Star Sports & Entertainment Network: Coming soon – KTTC News

    Safety concerns in Deep Ellum create apprehension as the entertainment district gains visitors – CBS News

    Safety Concerns Surge Amid Deep Ellum’s Booming Popularity and Growing Crowds

    Elisabeth Moss’ ‘Handmaid’s Tale’ Emmy chances, by the numbers – Yahoo

    Elisabeth Moss’ ‘Handmaid’s Tale’ Emmy chances, by the numbers – Yahoo

    ‘Gangs of London’ Producer Explains Season 3 Deaths, Hypes Season 4 – Citizen Tribune

    Gangs of London’ Producer Reveals Shocking Season 3 Deaths and Teases Exciting Season 4

  • General
  • Health
  • News

    Cracking the Code: Why China’s Economic Challenges Aren’t Shaking Markets, Unlike America’s” – Bloomberg

    Trump’s Narrow Window to Spread the Truth About Harris

    Trump’s Narrow Window to Spread the Truth About Harris

    Israel-Gaza war live updates: Hamas leader Ismail Haniyeh assassinated in Iran, group says

    Israel-Gaza war live updates: Hamas leader Ismail Haniyeh assassinated in Iran, group says

    PAP Boss to Niger Delta Youths, Stay Away from the Protest

    PAP Boss to Niger Delta Youths, Stay Away from the Protest

    Court Restricts Protests In Lagos To Freedom, Peace Park

    Court Restricts Protests In Lagos To Freedom, Peace Park

    Fans React to Jazz Jennings’ Inspiring Weight Loss Journey

    Fans React to Jazz Jennings’ Inspiring Weight Loss Journey

    Trending Tags

    • Trump Inauguration
    • United Stated
    • White House
    • Market Stories
    • Election Results
  • Science
  • Sports
  • Technology
    Tech Champions with Leo Bletnitsky of Healthy Technology Solutions – Buzz Media Group

    Meet Tech Champion Leo Bletnitsky of Healthy Technology Solutions

    Crypto’s true revolution is about humanity, not technology – Cointelegraph

    Crypto’s Real Revolution: Transforming Humanity Beyond Technology

    $1 Billion Problem: New Technology Could Save Your Daily Cup of Coffee – SciTechDaily

    The $1 Billion Challenge: How New Technology Could Rescue Your Daily Cup of Coffee

    Canada’s construction industry gets serious about investing in technology as pressure mounts to do more with less – Yahoo Finance

    Canada’s Construction Industry Accelerates Tech Investments to Overcome Growing Challenges and Boost Efficiency

    Workforce Technology Eases Staffing Shortages in Rural Health Care – AJMC

    Workforce Technology Eases Staffing Shortages in Rural Health Care – AJMC

    Get the lead out: Putting new at-home lead testing technology to the test | Denver7 Investigates – Denver7

    Putting the Latest At-Home Lead Testing Technology to the Ultimate Test

    Trending Tags

    • Nintendo Switch
    • CES 2017
    • Playstation 4 Pro
    • Mark Zuckerberg
No Result
View All Result
  • Home
  • Business
  • Entertainment
    ‘Puttin’ on the Ritz’: Civic Players bring ‘Young Frankenstein’ to life – Yahoo

    Civic Players Deliver a Hilarious and Unforgettable Performance of ‘Young Frankenstein

    ‘Wheel of Fortune’: Amputee Wins $60,000 After Breaking Incredible ‘Curse’ – Hastings Tribune

    Wheel of Fortune’ Amputee Breaks Incredible ‘Curse’ to Win $60,000!

    North Star Sports & Entertainment Network: Coming soon – KTTC News

    North Star Sports & Entertainment Network: Coming soon – KTTC News

    Safety concerns in Deep Ellum create apprehension as the entertainment district gains visitors – CBS News

    Safety Concerns Surge Amid Deep Ellum’s Booming Popularity and Growing Crowds

    Elisabeth Moss’ ‘Handmaid’s Tale’ Emmy chances, by the numbers – Yahoo

    Elisabeth Moss’ ‘Handmaid’s Tale’ Emmy chances, by the numbers – Yahoo

    ‘Gangs of London’ Producer Explains Season 3 Deaths, Hypes Season 4 – Citizen Tribune

    Gangs of London’ Producer Reveals Shocking Season 3 Deaths and Teases Exciting Season 4

  • General
  • Health
  • News

    Cracking the Code: Why China’s Economic Challenges Aren’t Shaking Markets, Unlike America’s” – Bloomberg

    Trump’s Narrow Window to Spread the Truth About Harris

    Trump’s Narrow Window to Spread the Truth About Harris

    Israel-Gaza war live updates: Hamas leader Ismail Haniyeh assassinated in Iran, group says

    Israel-Gaza war live updates: Hamas leader Ismail Haniyeh assassinated in Iran, group says

    PAP Boss to Niger Delta Youths, Stay Away from the Protest

    PAP Boss to Niger Delta Youths, Stay Away from the Protest

    Court Restricts Protests In Lagos To Freedom, Peace Park

    Court Restricts Protests In Lagos To Freedom, Peace Park

    Fans React to Jazz Jennings’ Inspiring Weight Loss Journey

    Fans React to Jazz Jennings’ Inspiring Weight Loss Journey

    Trending Tags

    • Trump Inauguration
    • United Stated
    • White House
    • Market Stories
    • Election Results
  • Science
  • Sports
  • Technology
    Tech Champions with Leo Bletnitsky of Healthy Technology Solutions – Buzz Media Group

    Meet Tech Champion Leo Bletnitsky of Healthy Technology Solutions

    Crypto’s true revolution is about humanity, not technology – Cointelegraph

    Crypto’s Real Revolution: Transforming Humanity Beyond Technology

    $1 Billion Problem: New Technology Could Save Your Daily Cup of Coffee – SciTechDaily

    The $1 Billion Challenge: How New Technology Could Rescue Your Daily Cup of Coffee

    Canada’s construction industry gets serious about investing in technology as pressure mounts to do more with less – Yahoo Finance

    Canada’s Construction Industry Accelerates Tech Investments to Overcome Growing Challenges and Boost Efficiency

    Workforce Technology Eases Staffing Shortages in Rural Health Care – AJMC

    Workforce Technology Eases Staffing Shortages in Rural Health Care – AJMC

    Get the lead out: Putting new at-home lead testing technology to the test | Denver7 Investigates – Denver7

    Putting the Latest At-Home Lead Testing Technology to the Ultimate Test

    Trending Tags

    • Nintendo Switch
    • CES 2017
    • Playstation 4 Pro
    • Mark Zuckerberg
No Result
View All Result
Earth-News
No Result
View All Result
Home Science

Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations

May 17, 2024
in Science
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations
Share on FacebookShare on Twitter

Main

Functional interactions among combinations of genetic elements underlie many natural and engineered phenotypes1,2,3, often involving higher-order (≥ 3-plex) combinations of coding4,5 or non-coding elements6,7,8,9. Experimentally testing higher-order combinations of genetic perturbations has been limited by throughput, with prior systematic analyses primarily performed in yeast10,11,12,13,14. In mammalian functional genomics, pooled screens15,16,17 using sequencing readouts have been limited to up to three genetic perturbations per cell when using RNA interference18 or CRISPR-Cas9 (ref. 19). Further multiplexing in Cas9-based pooled screening is challenging due to increasingly complex cloning schemes for large constructs encoding multiple guides expressed from separate promoters and length-dependent high recombination frequencies of lentiviral guide libraries20,21,22. Conceptually, it also remains unclear how to tractably survey the potentially vast combinatorial spaces for ≥3-plex perturbations.

Cas12a, a member of the type V CRISPR-Cas family, has been proposed as an alternative to Cas9 for genetic perturbations due to enhanced multiplexing capabilities. Cas12a harbors RNase activity, separable from its DNase activity, that can process a compact primary transcript expressed from a single promoter into multiple CRISPR RNAs (crRNAs)23,24. An array of multiple Cas12a crRNAs, each composed of a 19-nt direct repeat and a 19-to 23-nt spacer, can be encoded by a chemically synthesized oligo for single-step cloning into an expression vector25,26,27,28,29. Cas12a has been engineered for mammalian cell applications using its DNase activity to disrupt coding gene function using single or multiplexed crRNA constructs in individual well-based assays24,25,26,27,30,31 and in pooled sequencing screens28,29,31,32,33,34,35. However, extended multiplexing with fully DNase-competent Cas12a is expected to be constrained by genotoxicity from double-stranded DNA breaks in many biological contexts28,36,37,38,39,40,41. In principle, avoiding genotoxicity can be achieved by using DNase-dead Cas fusion proteins to control chromatin state and transcription, such as by DNase-dead Cas9 (dCas9)-based CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa)42,43,44. Moreover, CRISPRi is more efficient than DNA cutting at perturbing enhancers in pooled screens45,46,47, likely due to CRISPRi’s larger genomic window of activity via formation of repressive chromatin48. Thus, a DNase-dead Cas12a (dCas12a) functional genomics platform for multisite CRISPRi targeting would be highly desirable for testing the combinatorial functions of coding and non-coding genetic elements. However, no dCas12a-based pooled CRISPRi screening platform has been reported. Several studies have used dCas12a fusion proteins for CRISPRi in human cells in individual well-based assays, reporting either successful27,49,50,51 or unsuccessful52 repression of target genes. These dCas12a CRISPRi studies delivered crRNA plasmids by transient transfection rather than lentiviral transduction. Transient plasmid transfections express synthetic components at 10- to 1,000-fold higher than single-copy lentiviral integration of crRNA constructs, which is required in pooled screens to attribute cellular phenotypes to unique crRNA constructs by high-throughput sequencing15,16. Whether prior dCas12a CRISPRi constructs are sufficiently potent for pooled screens remains unclear.

In this study, we show that existing dCas12a CRISPRi fusion constructs function poorly when used with limiting doses of lentivirally delivered components, thus precluding their application in pooled screens. We engineered an Acidaminococcus Cas12a (AsCas12a) variant that incorporates a key mutation, R1226A, which enhances stability of the ribonucleoprotein–DNA complex in the form of a nicked DNA intermediate in vitro53,54. We show that in human cells, multiAsCas12a-KRAB fusion substantially improves CRISPRi activity in the setting of lentivirally delivered crRNA constructs, enabling use of 6-plex crRNA arrays in high-throughput pooled screens and up to 10-plex crRNA arrays in well-based assays. We use this combinatorial CRISPRi platform to efficiently discover enhancer elements and to test higher-order combinatorial perturbations of cis-regulatory elements. These results instantiate a group testing framework that enables efficient searches of potentially large combinatorial spaces of chromatin perturbations.

Results

Lentivirally delivered CRISPRi by dAsCas12a fusion proteins is hypoactive

We focused on building a CRISPRi functional genomics platform using AsCas12a, the only Cas12a ortholog with demonstrated success in pooled screens in mammalian cells28,29,31,32,33,34,55,56. A previous study reported using dAsCas12a for CRISPRi by plasmid transient transfection delivery of dAsCas12a-3xKRAB protein (harboring the E993A DNase-dead mutation) and crRNA in HEK 293T cells27. To test this construct in the setting of lentivirally delivered crRNA, we introduced dAsCas12a-3xKRAB by piggyBac transposition in K562 cells, followed by lentiviral transduction of single crRNA constructs targeting canonical (TTTV) or non-canonical30 protospacer adjacent motifs (PAMs) proximal to transcriptional start sites of four genes encoding for cell surface proteins, three of which (CD55, CD81 and B2M) have been successfully knocked down by dCas9-KRAB CRISPRi (Fig. 1a)57. Throughout this study we encoded crRNAs in a CROP-seq58 lentiviral vector previously optimized for pooled screens using DNase-active AsCas12a (ref. 29). We observed no expression change in any of the targeted genes (Fig. 1b and Supplementary Figs. 1 and 2). We confirmed the expression of dAsCas12a-3xKRAB by western blot (Supplementary Fig. 3) and by flow cytometry monitoring of the in-frame P2A-BFP (Supplementary Fig. 4a). We also observed this lack of CRISPRi activity for dAsCas12a-3xKRAB using lentivirally transduced crRNAs in C4-2B prostate cancer cells (Supplementary Fig. 4b). In contrast, transient co-transfection of dAsCas12a-3xKRAB and CD55-targeting crRNA plasmids shows modest CRISPRi knockdown in HEK 293T cells (Supplementary Fig. 5), consistent with prior work27. These findings indicate that the requirements for CRISPRi activity using dAsCas12a-3xKRAB with lentiviral crRNA constructs are distinct from those of plasmid transient transfection in HEK 293T cells27.

Fig. 1: dAsCas12a-KRAB variants are dose-limited and weak in CRISPRi activity when using lentivirally delivered crRNAs, despite incorporating state-of-the-art optimizations.figure 1

a, Schematic for assaying CRISPRi activity of AsCas12a constructs using lentivirally transduced single-plex or 3-plex crRNAs targeting cell surface marker genes assayed by antibody staining and flow cytometry. b, K562 cells constitutively expressing dAsCas12a-3xKRAB27 were lentivirally transduced with the indicated single crRNAs and assayed by flow cytometry 6 days after crRNA transduction. One of two biological replicates is shown; second replicate is shown in Supplementary Fig. 2. c, A panel of AsCas12a variants harboring combinations of mutations are tested using crCD55-4 and crCD81-1 using the fusion protein domain architecture shown. Both AsCas12a fusion protein and crRNA constructs are delivered by lentiviral transduction. D908A is a mutation in the RuvC catalytic triad that renders Cas12a DNase inactive24,54. Other mutations are described in detail in the main text. Shown are single-cell distributions of target gene expression assayed by flow cytometry 6 days after crRNA transduction for one of three independent replicates. Additional replicates and results for additional crRNA constructs (up to 3-plex crRNA constructs) are summarized in Supplementary Fig. 6a–c. d, Analysis of CD81 knockdown in cells lentivirally transduced with denAsCas12a-KRAB protein construct at multiplicity of infection (MOI) ~1 versus MOI ~5 while maintaining constant crRNA MOI ( 200 cells per replicate). c–e, Asterisks indicate P 
>>> Read full article>>>
Copyright for syndicated content belongs to the linked Source : Nature.com – https://www.nature.com/articles/s41587-024-02224-0

Tags: CRISPR-Casengineeredscience
Previous Post

Bidirectional epigenetic editing reveals hierarchies in gene regulation

Next Post

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex

Should You Buy Micron Technology Stock Before June 25? – The Motley Fool

Is Now the Perfect Moment to Invest in Micron Technology Before June 25?

June 21, 2025
Today’s NYT Connections: Sports Edition Hints and Answers for June 21, #271 – CNET

Today’s NYT Connections: Sports Edition Hints and Answers for June 21, #271 – CNET

June 21, 2025
Identifying ecological thresholds from functional traits for optimal ecosystem management – Nature

Unlocking Ecological Thresholds Through Functional Traits for Smarter Ecosystem Management

June 21, 2025
Speed-snap science: Solving for molecular details in a flash – Phys.org

Speed-Snap Science: Unlocking Molecular Secrets in a Flash

June 21, 2025
Fish Suffer Up to 22 Minutes of Intense Pain When Taken Out of Water – ScienceAlert

Fish Suffer Up to 22 Minutes of Intense Pain When Taken Out of Water – ScienceAlert

June 21, 2025
Brief Use of CGM May Enhance Lifestyle Awareness in T2D – Medscape

Brief Use of CGM May Enhance Lifestyle Awareness in T2D – Medscape

June 21, 2025
FIFA Club World Cup 2025: Botafogo stuns PSG, 1-0, in tournament’s first major upset – Yahoo Sports

Botafogo Stuns PSG with a Stunning 1-0 Triumph in FIFA Club World Cup 2025’s Biggest Upset

June 21, 2025
Study: Mass Deportations Would Cost California Economy $275 Billion, Decimate Critical Industries – University of California, Merced

Study: Mass Deportations Would Cost California Economy $275 Billion, Decimate Critical Industries – University of California, Merced

June 21, 2025
‘Puttin’ on the Ritz’: Civic Players bring ‘Young Frankenstein’ to life – Yahoo

Civic Players Deliver a Hilarious and Unforgettable Performance of ‘Young Frankenstein

June 21, 2025
Economics Student Studying Maternal Health Earns National Fellowship – Georgia State University News

Economics Student Studying Maternal Health Earns National Fellowship – Georgia State University News

June 21, 2025

Categories

Archives

June 2025
MTWTFSS
 1
2345678
9101112131415
16171819202122
23242526272829
30 
« May    
Earth-News.info

The Earth News is an independent English-language daily published Website from all around the World News

Browse by Category

  • Business (20,132)
  • Ecology (695)
  • Economy (709)
  • Entertainment (21,610)
  • General (15,490)
  • Health (9,750)
  • Lifestyle (715)
  • News (22,149)
  • People (712)
  • Politics (716)
  • Science (15,929)
  • Sports (21,206)
  • Technology (15,695)
  • World (689)

Recent News

Should You Buy Micron Technology Stock Before June 25? – The Motley Fool

Is Now the Perfect Moment to Invest in Micron Technology Before June 25?

June 21, 2025
Today’s NYT Connections: Sports Edition Hints and Answers for June 21, #271 – CNET

Today’s NYT Connections: Sports Edition Hints and Answers for June 21, #271 – CNET

June 21, 2025
  • About
  • Advertise
  • Privacy & Policy
  • Contact

© 2023 earth-news.info

No Result
View All Result

© 2023 earth-news.info

No Result
View All Result

© 2023 earth-news.info

Go to mobile version