DavidJonahGrunwaldDepartmentofHumanGenetics

UniversityofUtah

An Introduction to Genome Editing

Our characteristics are due to a combination of influences from both genes and environment

Some traits are strongly influenced by environment

Obesity (BMI ≥30 kg/m2)

Diagnosed diabetes

1994

1994

2000

2000

No Data <14.0% 14.0%–17.9% 18.0%–21.9% 22.0%–25.9% >26.0%

No Data <4.5% 4.5%–5.9% 6.0%–7.4% 7.5%–8.9% >9.0%

CDC’sDivisionofDiabetesTranslation.NationalDiabetesSurveillanceSystemhttp://www.cdc.gov/diabetes/statistics

2010

2010

Many adult attributes are determined upon formation of the fertilized egg

(set in wax, if not stone)

The “CRISPR revolution” is a technology that allows usto “edit” or modify the genes in human eggs/embryos

What kinds of changes can be attained by editing of the gene?

Whatkindsoftraits/characteristicscanbeinherited?

Whatkindsoftraitswithageneticbasisvaryamongindividualsofaspecies?

Whatdoesitmeanwhenscientistssayweallhave“thesamegenes”andyetclearlywedon’thave“identicalgenes”?Whatdoesageneencode?Howdogenesdifferamongindividuals?

HowmightchangingtheDNAsequenceofagenealtertraits?

Whatistherelationbetweenanindividualgeneandatrait?

Howdoweaccomplishgenomeediting?

Our Challenge:To understand how modifying the DNA sequence of an

egg/embryo might alter the future traits of that embryo

What kinds of traits/characteristics can be inherited?

Eachparenthastwosetsofchromosomes(twocompletesetsofgenes)andpassesonlyonecompletesetofgenestooffspring.

Adifferentassortmentofgenesistransmittedtoeachchild,sothateachchildinheritsauniquecombinationofgenesfrommother

What kinds of traits/characteristics can be inherited?

Ifweteaseoutallthevariationpresentinanaturalpopulationandjustcreategroupsofrelativelyinbredindividuals,wecanseemoreclearly(perhapsfrighteningly)whatcharacteristicscanbestablypassedfromparenttooffspring

Within a species we can isolate genetic variantsthat produce extreme variations in phenotype

What kinds of traits with a genetic basis vary among individuals of a species?

Clearly complicated traits, consisting of many parts have a genetic basis and can be inherited – nevertheless under normal circumstances, in outbred populations, we don’t see simple inheritance patterns of behavior.

What kinds of traits with a genetic basis vary among individuals of a species?

One reason is that many traits are “complex traits” governed by the actions of multiple genes working as an ensemble. Individual genes may contribute very very little to the overall trait

Control of blood pressure is a complex trait

Sandosh Padmanabhan, and Bina Joe Physiol Rev 2017;97:1469-1528

What does it mean when scientists say we all have “the same genes” and yet clearly we don’t have “identical genes”?

What is this “gene” and what does it really do?

We inherit one complete set of genes (called a genome) from each parent – in the form of 23 chromosomes – each

chromosome is a single very very very long molecule of DNA

Each DNA molecule is a linear array of only 4 letters! The key is the ordering of these letters.

The human genome contains DNA with a continuous linear arrangement of 3 billion letters (3,000,000,000)

What does it mean when scientists say we all have “the same genes” and yet clearly we don’t have “identical genes”?

What is this “gene” and what does it really do?

A gene is a portion of a DNA molecule with a particular function. It is information governing what protein (or RNA) might be made, under what conditions, when, where, how much

Protein coding

Control

Protein 1

Protein 2

Gene 1

Gene 2

There is punctuation indicating beginnings and ends of genes

The DNA sequence of a gene encodes a linear array of subcomponents (amino acids) of a protein. The gene sequence

is copied into mRNA and the mRNA is decoded

The DNA sequence of a gene encodes a linear array of subcomponents (amino acids) of a protein

The DNA sequence of a gene encodes a linear array of subcomponents (amino acids) of a protein –

This determines the structure, folding, and activity of a protein

Hemoglobin

A letter change in the DNA sequence can change one amino acid of a protein

having an effect on the structure, folding, or activity of a protein

Indeed a change in a single amino acid of a single protein is largely responsible for pigmentation differences in humans

Variation in our DNA sequences contributes to variation in the population. We all have the same genes – generally we make the

same proteins. But our genes encode proteins with slightly different

structures or activities or with differences in expression

▪ Wilt the “Stilt”

▪ Willie the ”Shu”

By and large, we don’t understandthe control of complex traits

Simple genetic diseases

variants in the sequence of 1 gene = 1 disease

Cystic Fibrosis

Neurofibromatosis

Huntington’s Corea

Some 4,000 discovered

Some traits are inherited as “simple Mendelian traits”

DNA sequence => protein => > > trait

Variant sequence => variant protein => > > variant trait

Goal of genome editing

Intended sequence => => > > intended trait

Fix a defective protein causing disease

Confer resistance

Alter behavior

Alter physical attribute

+donorDNA

**

HomologousRecombination

**

ReplacementandIntegrationSimultaneously

NonhomologousEndJoining

Smallinsertions/deletions

Breaks in the DNA molecule making up a chromosome are quickly recognized and repaired

Doudna and Charpentier (2014) Science 346,

CRISPR-Cas9 is a sequence-specific DNA break-inducer (nuclease)A True Revolution in Genome Engineering

CRISPRandCas9proteinsconstituteanadaptiveimmunitysystem,foundin~40%bacterialgenomesandmostarchaeal genomes

lysis

raresurvivors

ϕ859 or ϕ2972

Jinek et al. (2012) Science 337: 816

InStreptococcuspyogenes,threecomponentsnecessary:crRNA, tracrRNA,andCas9nuclease

Why the revolution?It is very simple to construct a highly specific CRISPR in the

laboratory

A variety of strategies for targeted genome editing

no template

NHEJ

HR

HDR

InjectCRISPRs+WTDNAsequence

intogol-/- zebrafish eggs

Analyze 48hpfG0embryosforpigmented cells

gol+/?gol-/-

Gene editing at golden

We can readily inject mammalian eggs with CRISPR components and donor DNA sequences

HowspecificistheCRISPRaction?

Whatistheefficiencyofmodification?

Doweunderstandthegeneticsunderlyingcomplextraits?

Open technical issues:

Questions?

ThankstoRayGesteland