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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
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