CRISPR Gene Editing: Methods and Protocols

356 Pages·2019·9.865 MB·English

by  Yonglun Luo

Methods in
Molecular Biology 1961
Yonglun Luo Editor
CRISPR
Gene Editing
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
CRISPR Gene Editing
Methods and Protocols
Edited by
Yonglun Luo
Department of Biomedicine, Aarhus University, Aarhus, Denmark
BGI-Shenzhen, Shenzhen, China
Editor
YonglunLuo
DepartmentofBiomedicine
AarhusUniversity
Aarhus,Denmark
BGI-Shenzhen
Shenzhen,China
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-9169-3 ISBN978-1-4939-9170-9 (eBook)
https://doi.org/10.1007/978-1-4939-9170-9
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Preface
When the series editor, Dr. John M. Walker, asked me approximately 1 year ago whether I
wasinterestedineditingabookonCRISPRtechnologies,Iimmediatelytookhisgenerous
offerandstartedmyfirstandchallengingeditorialjourney.Firstly,asajunior researcher,it
has always been a great honor and encouraging experience to be capable of contributing
withknowledgeandoutreachtoourscientificcommunity.Secondly,theparticularresearch
fieldandtechnologythatwewillbefocusingoninthisbookisoneofthemostfastgrowing
andimportantbreakthroughsduringthepastdecade.Andmostimportantly,asaresearcher
workingongeneeditingforover10years,Ideeplyrealizetheimportanceofhavingagood
serialofprotocols,experimentaltips,andnotestoincreasethesuccessrateandoutcomesof
scientificprojects.
WhenIfirststartedthe“PigandHealth”PhDprojectbackin2008,myobjectivewasto
recapitulate the pathogenesis of human diseases, e.g., breast cancer and diabetes, using
genetically tailored pig models. One major milestone of my PhD study was disrupting the
BRCA1 gene in primary porcine fibroblasts by homologous recombination and subse-
quently generating a BRCA1 knockout pig by somatic cell nuclear transfer. Generating a
gene knockout (KO) animal during the pre-TALENs and pre-CRISPR era was rather
technically challenging and time-consuming. Although I spent almost 3 years and finally
got my BRCA1 KO pig, I wish my project was conducted now rather than 10 years ago.
Zinc finger nucleases (ZFNs), known as the second generation of programmable DNA
nucleases, were already available during that period. But this technology was not very
broadlyadoptedbythescientificcommunity.OnemainreasonisthattheZFNtechnology
is relatively difficult for the design and generation, and there is a lack of user-friendly
protocolsandmethodsinstructingthegenerationandfunctionalvalidationofZFNs.
When the transcription activator-like effector (TALE) protein was engineered as a
programmable DNA endonuclease (TALEN) for gene editing, as compared to ZFNs,
TALEN-based gene editing was more rapidly applied by the whole scientific community.
One important driving force of the TALEN technology is conventional web tools and
protocols developed for TALEN vector design and construction. Using one the most
popular TALEN assembly methods developed by Daniel F. Voytas’s group, although one
hastoselect thedifferentmodularplasmidsfromalargestockofpremadeones,itismuch
easier whencomparedtoZFNstogenerateacoupleofTALENconstructions.
Sincethefirstproof-of-principlestudyofharnessingtheclusteredregularlyinterspaced
short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) for gene
editingin2012,ledbyJenniferDoudnaandEmmanuelleCharpentier,theCRISPR-Cas9-
based gene editing technology rapidly took over ZFNs and TALENs and was successfully
applied for genome editing in almost all cell types and organisms. To date, many Cas9
orthologs, different CRISPR systems from bacteria and archaea have been repurposed for
gene editing. And many more genetic manipulation tools have been built on top of the
CRISPR-Cas9 system, such as CRISPR-based gene activation, gene interference, base
editing, DNA methylation, and histone acetylation. Among all the already harnessed
CRISPR-Cas systems utilized for gene editing, the CRISPR-Cas9 system is still the most
extensivelydevelopedandbroadlyusedone.
v
vi Preface
The CRISPR-Cas9 technology is commonly known and described as simple, efficient,
andcost-effective.However,asuccessfulCRISPRgeneeditingexperiment/projectrequires
strategic planning and user-friendly guidelines to select the most suitable CRISPR-Cas
systemandtargetsiteswithhighactivityandspecificity.Also,howtoquantifytheCRISPR
gene editing activity (indel), how to efficiently deliver the CRISPR-Cas9 components into
target cells or tissues, and how to enrich and isolate gene-edited cells with desired genetic
modifications:theseareamongthemostfrequentlyaskedquestionsandexperimentswhen
conducting a CRISPR gene editing study. This book is intended to assist undergraduates,
graduates, and researchers with detailed guidelines and methods for the CRISPR gene
editingfield.
I would like to thank all the contributing authors at the front lines of developing
CRISPR technology and applications. Methods covering CRISPR gRNA design, CRISPR
delivery, CRISPR activity quantification (indel quantification), and examples of applying
CRISPR gene editing in human pluripotent stem cells, primary cells, gene therapy, and
genetic screening are included in this book. Without their contributions, this book could
neverhavebeenwritten.
MygratefulthanksalsotomycolleaguesfromtheLarsBolundInstituteofRegenerative
MedicineandtheDREAMteamfor theirassistanceineditingthebook.
Aarhus,Denmark YonglunLuo
Contents
Preface ..................................................................... v
Contributors................................................................. ix
PART I METHODS FOR CRISPR-GRNA DESIGN AND QUANTIFICATION
OF ACTIVITY
1 CRISPR-gRNADesign.................................................. 3
MariaPallare`sMasmitj`a,NastassiaKn¨odlseder,andMarcGu¨ell
2 TrackingCRISPR’sFootprints ........................................... 13
LinLinandYonglunLuo
3 RapidQuantitativeEvaluationofCRISPRGenomeEditingbyTIDE
andTIDER ............................................................ 29
EvaKarinaBrinkmanandBasvanSteensel
4 FastandQuantitativeIdentificationofExVivoPreciseGenome
Targeting-InducedIndelEventsbyIDAA ................................. 45
SaskiaK¨onig,ZhangYang,HansHeughWandall,ClaudioMussolino,
andEricPaulBennett
5 FunctionalEvaluationofCRISPRActivitybytheDual-Fluorescent
SurrogateSystem:C-Check .............................................. 67
LinLinandYonglunLuo
PART II METHODS FOR CRISPR DELIVERY
6 CRISPR-Cas9DeliverybyArtificialVirus(RRPHC)........................ 81
SuleixinYang,QinjieWu,YuquanWei,andChangyangGong
7 ProductionandValidationofLentiviralVectorsforCRISPR/Cas9Delivery ... 93
LauraBarrettRyø,EmilAagaardThomsen,andJacobGiehmMikkelsen
8 RapidandSimpleScreeningofCRISPRGuideRNAs(gRNAs)
inCulturedCellsUsingAdeno-AssociatedViral(AAV)Vectors............... 111
JuliaFakhiri,ManuelaNickl,andDirkGrimm
9 Electroporation-BasedCRISPR/Cas9GeneEditingUsingCas9
ProteinandChemicallyModifiedsgRNAs ................................. 127
AndersLaustsenandRasmusO.Bak
PART III CRISPR GENE EDITING IN HUMAN IPSCS
10 EfficientGeneEditingofHumanInducedPluripotentStemCells
UsingCRISPR/Cas9 ................................................... 137
SaniyeYumlu,SanumBashir,Ju¨rgenStumm,andRalfKu¨hn
vii
viii Contents
11 EditingtheGenomeofHumanInducedPluripotentStemCellsUsing
CRISPR/Cas9RibonucleoproteinComplexes.............................. 153
MichaelaBruntraeger,MegByrne,KathleenLong,
andAndrewR.Bassett
12 ConditionalGeneKnockoutinHumanCellswithInducible
CRISPR/Cas9 ......................................................... 185
KirstenE.Snijders,JamesD.Cooper,LudovicVallier,
andAlessandroBertero
PART IV CRISPR GENE EDITING IN OTHER CELL TYPES
13 CRISPR/Cas9asaGenomeEditingToolforTargetedGeneIntegration
inCHOCells .......................................................... 213
DariaSergeeva,JoseManuelCamacho-Zaragoza,JaeSeongLee,
andHeleneFaustrupKildegaard
14 RapidandEfficientGeneDeletionbyCRISPR/Cas9 ....................... 233
SigneNeldeborg,LinLin,MagnusStougaard,andYonglunLuo
15 GenomeEditinginMice ................................................ 249
LisbethAhmHansenandErnst-MartinFu¨chtbauer
16 CRISPR/Cas9-MediatedGeneTagging:AStep-by-StepProtocol............ 255
XiXiang,ConghuiLi,XiChen,HongweiDou,YongLi,
XiuqingZhang,andYonglunLuo
17 GeneEditinginPrimaryCellsofCattleandPig............................ 271
PetraVochozkova,KilianSimmet,Eva-MariaJemiller,
AnnegretWu¨nsch,andNikolaiKlymiuk
PART V CRISPR GENE THERAPYAND SCREENING
18 TowardInVivoGeneTherapyUsingCRISPR............................. 293
KristianAlsbjergSkipperandJacobGiehmMikkelsen
19 CRISPRGeneTherapyoftheEye:TargetedKnockoutofVegfa
inMouseRetinabyLentiviralDelivery .................................... 307
AndreasHolmgaard,SidselAlsing,AnneLouiseAskou,
andThomasJ.Corydon
20 InVivoEditingoftheAdultMouseLiverUsingCRISPR/Cas9
andHydrodynamicTailVeinInjection .................................... 329
FrancescoNiola,FrederikDagnæs-Hansen,andMortenFr¨odin
21 CRISPR-BasedLentiviralKnockoutLibrariesforFunctionalGenomic
ScreeningandIdentificationofPhenotype-RelatedGenes ................... 343
EmilAagaardThomsenandJacobGiehmMikkelsen
Index ...................................................................... 359
Contributors
SIDSELALSING (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark
ANNE LOUISE ASKOU (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark
RASMUSO.BAK (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark;
AarhusInstituteofAdvancedStudies(AIAS),AarhusUniversity,Aarhus,Denmark
SANUMBASHIR (cid:1) Max-Delbru¨ck-Centrumfu¨rMolekulareMedizin,Berlin,Germany;Berlin
InstituteofHealth,Berlin,Germany
ANDREWR.BASSETT (cid:1) WellcomeSangerInstitute,WellcomeGenomeCampus,Hinxton,
Cambridge,UK
ERICPAULBENNETT (cid:1) FacultyofHealthSciences,DepartmentofOdontology,Copenhagen
Center forGlycomics(CCG),UniversityofCopenhagen,Copenhagen,Denmark
ALESSANDROBERTERO (cid:1) WellcomeTrust-MRCStemCellInstitute,AnneMcLaren
Laboratory,UniversityofCambridge,Cambridge,UK;DepartmentofSurgery,University
ofCambridge,Cambridge,UK;DepartmentofPathology,UniversityofWashington,
Seattle,WA,USA
EVAKARINABRINKMAN (cid:1) DivisionofGeneRegulationandOncodeInstitute,Netherlands
CancerInstitute,Amsterdam,TheNetherlands
MICHAELABRUNTRAEGER (cid:1) WellcomeSangerInstitute,WellcomeGenomeCampus,Hinxton,
Cambridge,UK
MEGBYRNE (cid:1) WellcomeSangerInstitute,WellcomeGenomeCampus,Hinxton,Cambridge,
UK
JOSEMANUELCAMACHO-ZARAGOZA (cid:1) TheNovoNordiskFoundationCenter for
Biosustainability,TechnicalUniversityofDenmark,Kgs.Lyngby,Denmark
XICHEN (cid:1) BGI-Shenzhen,Shenzhen,China
JAMESD.COOPER (cid:1) WellcomeTrust-MRCStemCellInstitute,AnneMcLarenLaboratory,
UniversityofCambridge,Cambridge,UK;DivisionofCardiovascularMedicine,
UniversityofCambridge,Cambridge,UK
THOMASJ.CORYDON (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark;
DepartmentofOphthalmology,AarhusUniversityHospital,Aarhus,Denmark
FREDERIK DAGNÆS-HANSEN (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,
Denmark
HONGWEI DOU (cid:1) BGI-Qingdao,Qingdao,China
JULIAFAKHIRI (cid:1) DepartmentofInfectiousDiseases/Virology,HeidelbergUniversityHospital,
ClusterofExcellenceCellNetworks,Heidelberg,Germany;BioQuantCenter,Universityof
Heidelberg,Heidelberg,Germany
MORTENFRO¨DIN (cid:1) BiotechResearchandInnovationCentre(BRIC),Universityof
Copenhagen,Copenhagen,Denmark
ERNST-MARTINFU¨CHTBAUER (cid:1) DepartmentofMolecularBiologyandGenetics,Aarhus,
Denmark
CHANGYANGGONG (cid:1) StateKeyLaboratoryofBiotherapyandCancerCenter,WestChina
Hospital,SichuanUniversity,Chengdu,China
ix
x Contributors
DIRKGRIMM (cid:1) DepartmentofInfectiousDiseases/Virology,HeidelbergUniversityHospital,
ClusterofExcellenceCellNetworks,Heidelberg,Germany;BioQuantCenter,Universityof
Heidelberg,Heidelberg,Germany;GermanCenterforInfectionResearch(DZIF),Partner
SiteHeidelberg,Heidelberg,Germany
MARCGU¨ELL (cid:1) DepartmentofExperimentalandHealthSciences,UniversitatPompeu
Fabra,Barcelona,Spain
LISBETHAHMHANSEN (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark
ANDREASHOLMGAARD (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark
EVA-MARIAJEMILLER (cid:1) InstituteforMolecularAnimalBreedingandBiotechnology,LMU
Munich,Munich,Germany
HELENEFAUSTRUPKILDEGAARD (cid:1) TheNovoNordiskFoundationCenterforBiosustainability,
TechnicalUniversityofDenmark,Kgs.Lyngby,Denmark
NIKOLAIKLYMIUK (cid:1) InstituteforMolecularAnimalBreedingandBiotechnology,LMU
Munich,Munich,Germany
NASTASSIAKNO¨DLSEDER (cid:1) DepartmentofExperimentalandHealthSciences,Universitat
PompeuFabra,Barcelona,Spain
SASKIAKO¨NIG (cid:1) MedicalCenter—UniversityofFreiburg,InstituteforTransfusionMedicine
andGeneTherapyandCenter forChronicImmunodeficiencyatCenter forTranslational
CellResearch(ZTZ),Freiburg,Germany
RALFKU¨HN (cid:1) Max-Delbru¨ck-Centrumfu¨rMolekulareMedizin,Berlin,Germany;Berlin
InstituteofHealth,Berlin,Germany
ANDERSLAUSTSEN (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark
JAESEONGLEE (cid:1) DepartmentofMolecularScienceandTechnology,AjouUniversity,Suwon,
RepublicofKorea
CONGHUI LI (cid:1) BGI-Qingdao,Qingdao,China
YONGLI (cid:1) BGI-Shenzhen,Shenzhen,China
LINLIN (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark
KATHLEENLONG (cid:1) WellcomeSangerInstitute,WellcomeGenomeCampus,Hinxton,
Cambridge,UK
YONGLUNLUO (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark;BGI-
Shenzhen,Shenzhen,China;GuangdongProvincialKeyLaboratoryofGenomeReadand
Write,Shenzhen,China;BGI-Qingdao,Qingdao,China
JACOBGIEHMMIKKELSEN (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,
Denmark
CLAUDIOMUSSOLINO (cid:1) MedicalCenter—UniversityofFreiburg,InstituteforTransfusion
MedicineandGeneTherapyandCenter forChronicImmunodeficiencyatCenter for
TranslationalCellResearch(ZTZ),Freiburg,Germany
SIGNE NELDEBORG (cid:1) DepartmentofClinicalMedicine,AarhusUniversity,Aarhus,
Denmark
MANUELA NICKL (cid:1) DepartmentofInfectiousDiseases/Virology,HeidelbergUniversity
Hospital,ClusterofExcellenceCellNetworks,Heidelberg,Germany;BioQuantCenter,
UniversityofHeidelberg,Heidelberg,Germany;GermanCenter forInfectionResearch
(DZIF),PartnerSiteHeidelberg,Heidelberg,Germany
FRANCESCONIOLA (cid:1) BiotechResearchandInnovationCentre(BRIC),Universityof
Copenhagen,Copenhagen,Denmark
MARIAPALLARE`SMASMITJA` (cid:1) DepartmentofExperimentalandHealthSciences,Universitat
PompeuFabra,Barcelona,Spain
LAURA BARRETTRYØ (cid:1) DepartmentofBiomedicine,AarhusUniversity,Aarhus,Denmark