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