BIOL335: Functional genomics
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Transcript of BIOL335: Functional genomics
Functional Annotation of Genomes
Paul Gardner
March 31, 2015
Paul Gardner Functional Annotation of Genomes
Discussion
I How can we identify functional regions within genomes?
Paul Gardner Functional Annotation of Genomes
Loss-of-function methods: knock-outs & knock-downs
I Two approaches for testing gene function with LOF methods:I Knocking out genes by deletion or disruption of functional
regionsI Knocking down genes by introducing antisense RNA (i.e.
RNAi)
I Assay the impact of the knock out/down
Paul Gardner Functional Annotation of Genomes
Can we knock genes out on a large scale?
I Idea 1: generate populations of mutants with transposableelements.
Hutchison III et al. (1999) Global Transposon Mutagenesis and a Minimal Mycoplasma Genome. Science
Paul Gardner Functional Annotation of Genomes
The Tn5 transposon
I ∼ 5700 BP in total
I Insertion seqs: IS50L andIS50R are ∼ 1533 BP(inverted repeats)
I Kanomycin, streptomycinand bleomycin antibioticresistance genes
I 476aa transposase (Tnp),catalyses transposition
I Tnp in complex withoutside ends (OE) DNAfragments
Reznikoff (2003) Tn5 as a model for understanding DNA transposition. Molecular Microbiology.
Paul Gardner Functional Annotation of Genomes
Tn-seq
I Transposons are represented by pink lines, sequencingadaptors by blue, genomic DNA by black and PCR primers bygreen.
Barquist, Boinett & Cain (2013) Approaches to querying bacterial genomes with transposon-insertion sequencing.RNA Biology
Paul Gardner Functional Annotation of Genomes
Tn-seq (called TraDIS in the UK)
I Electrotransformation with Tn5-derivedtransposon/transposase complex containing a kanamycin-resistance gene
I > 10 transformations per batch at 42, 000− 146, 000 mutantsper batch; 13 batches → 1.1 million mutants
I Apply some competitive growth conditions
I Short read sequencing with transposon specific primers
Langridge et al. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutantsGenome research
Paul Gardner Functional Annotation of Genomes
Tn-seq
I Insertion Index:count the numberof observed insertsin each gene,divide by thelength
Langridge et al. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutantsGenome research
Paul Gardner Functional Annotation of Genomes
Tn-seq on RNA in Salmonella
05101520253035
RNAseq
reads
020406080100
TraDIS
reads
994000 994100 994200 994300 994400
genome coordinate
dsrA
putative transcript yodD
Paul Gardner Functional Annotation of Genomes
Comparative Tn-seq
Barquist L, Langridge GC, Turner DJ, Phan MD, Turner AK, Bateman A, Parkhill J, Wain J, Gardner PP (2013)A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium. NucleicAcids Research.
Paul Gardner Functional Annotation of Genomes
Comparative Tn-seq
(A) the overlap of all genes (outer circles) and required genes(inner circles) Black numbers refer to all genes, whitenumbers to required genes.
(B) the overlap of all required genes between S. Typhimurium, S.Typhi and E. coli.
Barquist L, Langridge GC, Turner DJ, Phan MD, Turner AK, Bateman A, Parkhill J, Wain J, Gardner PP (2013)A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium. NucleicAcids Research.
Paul Gardner Functional Annotation of Genomes
Advantages and disadvantages
I Advantages:I It’s cool!I Relatively quick to prepare libraries and sequenceI The analysis is beautiful
I Disadvantages:I Paralogs are a pain (they compensate for each other)I DNA binding proteins (e.g. H-NS) may inhibit insertionI Quite a bit of workI Only identify the genes that are essential for the conditions
that were assayed
Paul Gardner Functional Annotation of Genomes
What’s more useful to know, essential or functional?
I How could these be distinguished?
I Are essential genes guaranteed to be functional? Arefunctional genes essential?
Paul Gardner Functional Annotation of Genomes
Knockouts in Eukaryotes: cool kids now use CRISPR/CAS
I Idea 3: systematic KOs at targeted loci using CRISPR/CAS.
Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science.Cong et al. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science.Mali et al. (2013) RNA-Guided Human Genome Engineering via Cas9. Science.
Paul Gardner Functional Annotation of Genomes
CRISPR/CAS background
I CRISPR-Cas:clustered, regularlyinterspaced shortpalindromic repeats
I Adaptive immunityfor 1
2 bacteria & mostarchaea
Westra, Buckling & Fineran (2014) CRISPRCas systems: beyond adaptive immunity. Nature Reviews Microbiology.
Paul Gardner Functional Annotation of Genomes
Knockouts in Eukaryotes: CRISPR-Cas
Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science.
Paul Gardner Functional Annotation of Genomes
Knockouts in Eukaryotes: CRISPR-Cas
I KO library of 18,080 genes with 64,751 guide seqs
I Used to identify genes essential for cell viability in pluripotentstem cells & in tumor tissue
I Screened for KOs that conferred resistance to vemurafenib(oncogene inhibitor, PLX, B-Raf enzyme inhibitor) in amelanoma model
Paul Gardner Functional Annotation of Genomes
Knockouts in Eukaryotes: CRISPR-Cas - essential genes
I Ranked genes by “depletion” of KOs
I Grouped genes by functional gene category & identified themost depleted categories
A375: melanoma HUES62: stem cell
Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science.
Paul Gardner Functional Annotation of Genomes
Knockouts in Eukaryotes: CRISPR-Cas - melanoma
I 5 genes whose KO confer resistance to PLX in a melanoma
I Include neurofibromin 2 (NF2), Cullin 3 E3 ligase (CUL3),and members of the STAGA histone acetyltransferase complex(TADA1 and TADA2B)
Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science.
Paul Gardner Functional Annotation of Genomes
Potential applications of CRISPR-Cas are very exciting
I Lots of basic science applicationsI Anti-viral and cancer therapiesI Correct genetic abnormalitiesI Engineered Cas9 used to alter methylation (and regulation)I Many “lifestyle” applications are possibleI Still very contentious (patent disbutes, Wikipedia edit wars,
etc.)
Paul Gardner Functional Annotation of Genomes
A call for a moratorium on CRISPR-Cas
Paul Gardner Functional Annotation of Genomes
Knockdowns used to be used
I Idea 2 (for those that arecounting)
I Based on RNAi andmicroRNAs
I 5,000 uniqueshRNA-expressinglentiviruses that target1,028 (≈ 5%) humangenes
I ≈ 100 candidate regulatorsof mitotic progression andproliferation
Mofatt et al. (2006) A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed ViralHigh-Content Screen. Cell.
Paul Gardner Functional Annotation of Genomes
Some conclusions
I Loss of function methods are a powerful tool for identifyinggene function (both coding and non-coding)
I The technology is evolving rapidly
I Screening large numbers of phenotypes is now a majorbottleneck & consistently linking these datasets is an openproblem
I A useful independent dimension to “traditional” measures ofgene function e.g. evolutionary conservation and expression
Paul Gardner Functional Annotation of Genomes
Relevant reading
I Reviews:I Barquist, Boinett & Cain (2013) Approaches to querying
bacterial genomes with transposon-insertion sequencing. RNABiology
I Westra, Buckling & Fineran (2014) CRISPRCas systems:beyond adaptive immunity. Nature Reviews Microbiology.
I Application:I Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout
Screening in Human Cells. Science.I CRISPR special collection:
www.sciencemag.org/site/extra/crispr
Paul Gardner Functional Annotation of Genomes
The End
Paul Gardner Functional Annotation of Genomes