Spacer2PAM: A computational framework for identification of functional PAM sequences for endogenous CRISPR systems

RNA-guided nucleases from clustered regularly interspaced short palindromic repeats (CRISPR) systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR systems in many bacteria and archaea are particularly attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR effectors. However, each CRISPR system recognizes a unique set of PAM sequences, which requires extensive screening of randomized DNA libraries. This challenge makes it difficult to develop endogenous CRISPR systems, especially in organisms that are slow-growing or have transformation idiosyncrasies. To address this limitation, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to identify potential PAM sequences for any CRISPR system given its corresponding CRISPR array as input. Spacer2PAM can be used in Quick mode to generate a single PAM prediction that is likely to be functional or in Comprehensive mode to inform targeted, unpooled PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-predicted PAM library for the type I-B CRISPR system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR systems for industrial biotechnology and synthetic biology.

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Faculty Opinions recommendation of Selection of chromosomal DNA libraries using a multiplex CRISPR system.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718538751.793499318 ◽

2014 ◽

Author(s):

Corey Nislow

Keyword(s):

Chromosomal Dna ◽

Crispr System ◽

Dna Libraries ◽

Selection Of

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Progress in zinc finger nuclease engineering for targeted genome modification

Hereditas (Beijing) ◽

10.3724/sp.j.1005.2011.00665 ◽

2011 ◽

Vol 33 (7) ◽

pp. 665-683 ◽

Cited By ~ 6

Author(s):

An XIAO ◽

Ying-Ying HU ◽

Wei-Ye WANG ◽

Zhi-Peng YANG ◽

Zhan-Xiang WANG ◽

...

Keyword(s):

Zinc Finger ◽

Zinc Finger Nuclease ◽

Genome Modification ◽

Targeted Genome Modification

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Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

mBio ◽

10.1128/mbio.01112-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 21

Author(s):

Pierre Boudry ◽

Ekaterina Semenova ◽

Marc Monot ◽

Kirill A. Datsenko ◽

Anna Lopatina ◽

...

Keyword(s):

Clostridium Difficile ◽

Type I ◽

Human Pathogen ◽

Range Analysis ◽

Data Set ◽

Content Type ◽

Crispr System ◽

Nosocomial Diarrhea ◽

Active Expression ◽

Foreign Dna

ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. IMPORTANCE Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide. To survive in bacteriophage-rich gut communities, enteropathogens must develop efficient systems for defense against foreign DNA elements. CRISPR-Cas systems have recently taken center stage among various anti-invader bacterial defense systems. We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages. These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.

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Implementation of the Omega (ω) Index to detect large-scale systematic cheating

10.35542/osf.io/exwkp ◽

2019 ◽

Author(s):

Alvin Vista

Keyword(s):

Standardized Testing ◽

Large Scale ◽

Type I Error ◽

R Package ◽

Statistical Testing ◽

System Level ◽

Control Group ◽

Type I ◽

Data Contamination ◽

Cheating Detection

Cheating detection is an important issue in standardized testing, especially in large-scale settings. Statistical approaches are often computationally intensive and require specialised software to conduct. We present a two-stage approach that quickly filters suspected groups using statistical testing on an IRT-based answer-copying index. We also present an approach to mitigate data contamination and improve the performance of the index. The computation of the index was implemented through a modified version of an open source R package, thus enabling wider access to the method. Using data from PIRLS 2011 (N=64,232) we conduct a simulation to demonstrate our approach. Type I error was well-controlled and no control group was falsely flagged for cheating, while 16 (combined n=12,569) of the 18 (combined n=14,149) simulated groups were detected. Implications for system-level cheating detection and further improvements of the approach were discussed.

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Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

Genetics ◽

10.1534/genetics.110.117002 ◽

2010 ◽

Vol 186 (2) ◽

pp. 451-459 ◽

Cited By ~ 195

Author(s):

Iara D. Carbery ◽

Diana Ji ◽

Anne Harrington ◽

Victoria Brown ◽

Edward J. Weinstein ◽

...

Keyword(s):

Zinc Finger ◽

Zinc Finger Nucleases ◽

Genome Modification ◽

Targeted Genome Modification

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DECENT: differential expression with capture efficiency adjustmeNT for single-cell RNA-seq data

Bioinformatics ◽

10.1093/bioinformatics/btz453 ◽

2019 ◽

Vol 35 (24) ◽

pp. 5155-5162 ◽

Cited By ~ 10

Author(s):

Chengzhong Ye ◽

Terence P Speed ◽

Agus Salim

Keyword(s):

Single Cell ◽

Differential Expression ◽

Type I Error ◽

R Package ◽

Supplementary Information ◽

Type I ◽

Common Phenomenon ◽

Rna Seq ◽

Capture Process ◽

Technological Platforms

Abstract Motivation Dropout is a common phenomenon in single-cell RNA-seq (scRNA-seq) data, and when left unaddressed it affects the validity of the statistical analyses. Despite this, few current methods for differential expression (DE) analysis of scRNA-seq data explicitly model the process that gives rise to the dropout events. We develop DECENT, a method for DE analysis of scRNA-seq data that explicitly and accurately models the molecule capture process in scRNA-seq experiments. Results We show that DECENT demonstrates improved DE performance over existing DE methods that do not explicitly model dropout. This improvement is consistently observed across several public scRNA-seq datasets generated using different technological platforms. The gain in improvement is especially large when the capture process is overdispersed. DECENT maintains type I error well while achieving better sensitivity. Its performance without spike-ins is almost as good as when spike-ins are used to calibrate the capture model. Availability and implementation The method is implemented as a publicly available R package available from https://github.com/cz-ye/DECENT. Supplementary information Supplementary data are available at Bioinformatics online.

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Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

mBio ◽

10.1128/mbio.01751-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 31

Author(s):

April Pawluk ◽

Megha Shah ◽

Marios Mejdani ◽

Charles Calmettes ◽

Trevor F. Moraes ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcriptional Repressor ◽

Mobile Genetic Elements ◽

Type I ◽

Genetic Studies ◽

Genetic Elements ◽

Crispr System ◽

Mechanistic Insight ◽

Resistance To Invasion ◽

Insight Into

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system.

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Integrating the evidence from evidence factors in observational studies

Biometrika ◽

10.1093/biomet/asz003 ◽

2019 ◽

Vol 106 (2) ◽

pp. 353-367 ◽

Cited By ~ 3

Author(s):

B Karmakar ◽

B French ◽

D S Small

Keyword(s):

Sensitivity Analysis ◽

Observational Study ◽

Error Rate ◽

Type I Error ◽

R Package ◽

Sensitivity Analyses ◽

Bahadur Efficiency ◽

Type I ◽

Familywise Error Rate ◽

Risk Of Cancer

Summary A sensitivity analysis for an observational study assesses how much bias, due to nonrandom assignment of treatment, would be necessary to change the conclusions of an analysis that assumes treatment assignment was effectively random. The evidence for a treatment effect can be strengthened if two different analyses, which could be affected by different types of biases, are both somewhat insensitive to bias. The finding from the observational study is then said to be replicated. Evidence factors allow for two independent analyses to be constructed from the same dataset. When combining the evidence factors, the Type I error rate must be controlled to obtain valid inference. A powerful method is developed for controlling the familywise error rate for sensitivity analyses with evidence factors. It is shown that the Bahadur efficiency of sensitivity analysis for the combined evidence is greater than for either evidence factor alone. The proposed methods are illustrated through a study of the effect of radiation exposure on the risk of cancer. An R package, evidenceFactors, is available from CRAN to implement the methods of the paper.

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