CRISPR-Cas Methods

288 Pages·2020·8.033 MB·English

by  M. Tofazzal Islam, Pankaj K. Bhowmik, Kutubuddin A. Molla

M. Tofazzal Islam
Pankaj K. Bhowmik
Kutubuddin A. Molla Editors
CRISPR-Cas
Methods
S P H
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CRISPR-Cas Methods
Edited by
M. Tofazzal Islam
Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman
Agricultural University, Gazipur, Bangladesh
Pankaj K. Bhowmik
Department of Aquatic and Crop Resource Development, National Research Council of Canada,
SASKATOON, SK, Canada
Kutubuddin A. Molla
Crop Improvement Division, ICAR-National Rice Research Institute, Cuttack, Odisha, India
Editors
M.TofazzalIslam PankajK.Bhowmik
InstituteofBiotechnologyandGenetic DepartmentofAquaticandCropResourceDevelopment
Engineering(IBGE) NationalResearchCouncilofCanada
BangabandhuSheikhMujibur SASKATOON,SK,Canada
RahmanAgriculturalUniversity
Gazipur,Bangladesh
KutubuddinA.Molla
CropImprovementDivision
ICAR-NationalRiceResearchInstitute
Cuttack,Odisha,India
ISSN1949-2448 ISSN1949-2456 (electronic)
SpringerProtocolsHandbooks
ISBN978-1-0716-0615-5 ISBN978-1-0716-0616-2 (eBook)
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Foreword
Sometimes in February 2016, I got in touch with Prof. Tofazzal Islam—an editor of this
book—aboutapplyingpathogenomicstoadiseaseepidemicthatjustbrokeoutinhisnative
country of Bangladesh. This story has been told over and over, and our perspective is
documented in an article we wrote a few months ago after an emotional visit to affected
farmsinBangladesh(Kamoun,S.,Talbot,N.J.,andIslam,M.T.2019.PLOSBiology,17:
e3000302).However,whatIdoubtisthateitheroneofusexpectedatthetimethechainof
events that have transformed our career paths and ultimately led to this book. What
happenedisthattheblastfungus—theculpritofthatdreadfuloutbreak—notonlyinfected
the wheat crop but also ended up redirecting our research activities towards investigating
this formidable foe. That journey took us to view CRISPR-Cas9 geneediting ofwheat for
blast disease resistance as a particularly promising approach to deliver solutions to this
problem.
How to manage destructive plant pathogens such as the wheat blast fungus? Genetics
has to be part of the answer. Crop germplasm screens and breeding towards disease
resistance is valuable but can be slow with limited potential. Biotechnology is a promising
alternativethathasyettodeliveritsfullpotentialtoplanthealthproblemslikewheatblast.
One such technology is gene editing or bioediting. Geneticists have long dreamed about
editing their organisms’ genomes as one would edit text on a computer with a word
processor. The dream has started to become a reality when CRISPR-Cas9 took the
biologicalworldbystormjustafewyearsagoin2013.Othergeneeditingmethods,notably
zinc-finger nucleases and TALENs,were availableat thetimebutCRISPR-Cas9 democra-
tized gene editing in the biological sciences. It has made gene editing relatively easy and
accessibletoprettymuchanylaboratorywithbasicmolecularbiologyskills.Thebreadthof
topicsandauthorsthatProf.Islamandhisco-editorsDr.PankajK.BhowmikandDr.Ku-
tubuddinA.Mollahavelinedupinthisbookisagreatexampleoftherangeofdevelopments
andapplicationsofthistechnologyanditspotentialtoimpactplanthealthandmanyother
areasofbiology.
WhyanotherbookonCRISPR?Theseriesofchaptersreflecttheuniqueperspectiveof
theauthorsandtheiraspirationtoseeCRISPR-Cas9bioeditingappliedtotheimprovement
ofcropsandthehumanconditioningeneral.Myhopeisthatthebookwouldguideanew
generationofplantgeneeditorsfromallovertheworld.Ihopeitwouldinspireearlycareer
scientists, particularly from developing countries, to embrace gene editing technology and
forgetheirownpathinthisrapidlyexpandingareaofgenetics.
TheSainsburyLaboratory,Norwich,UK SophienKamoun
v
Preface
Precisely altering genetic sequences—in almost any kind of living cells including those of
humans,atmuchhigheraccuracyandefficiencythaneverbeforebecamepossibleonlyafter
the recent discovery of genome editing technologies like zinc-finger nucleases (ZFNs),
transcriptional activator-like effector nucleases (TALENs), and clustered regularly inter-
spacedshortpalindromicrepeats(CRISPR)-associatednucleases(Cas).Thosetechnologies
allowustomakesmallchangestoaDNAsequencesataknownlocation.ComparedtoZFN
and TALEN, the CRISPR-Cas technology is faster, cheaper, and more user-friendly.
Although CRISPR-Cas system was originally identified as an adaptive immune system
from bacteria, its repurposing into versatile RNA-guided, DNA-targeting platforms revo-
lutionizedbasicbiology,agriculture,andmedicine.
Since discovery, the CRISPR-Cas9 technology has attracted widespread attention of
biologists, and its application has expanded dramatically in many areas including plant,
microbial,andanimalsciences.Overthelast6years,theCRISPR-Cassystemshavebecome
a powerful toolbox for genetic manipulation on the basis of simpler RNA-guided DNA
recognition.AsmallguideRNA(gRNA)directstheprotein,Cas,toatargetedlocustobind
with and make a double-strand break. Due to tremendous improvement in the methodol-
ogies, the CRISPR-Cas toolbox is now a useful technology for crop improvement, curing
geneticdiseases,andengineeringdesirablegenetictraits,aswellasnewapplicationslikelive-
cell imaging, high-throughput functional genomic screens, transcriptional regulation, epi-
genome modification, gene-drive, and point-of-care diagnostic. The diversity, modularity,
andefficacyofCRISPR-Cassystemsaredrivingabiotechnologicalrevolution.
OneoftheuniquefeaturesoftheCRISPR-CassystemisthecreationofDNAdouble-
strand breaks (DSBs) at target loci. This feature can be used to introduce a variety of
modifications in the genome of any target organism, including crop plants. Immediately
after the DSBs, two main DNA repair pathways naturally become functional—
(1) non-homologous end joining (NHEJ) and (2) homology-directed repair (HDR). In
higher eukaryotes, NHEJ is predominant and error-prone. During NHEJ-mediated DSB
repair,thecellularmachineryerroneouslyincorporatessmallinsertionordeletions(indel)at
thecutpoint.Asaresult,theopenreadingframeisdisrupted,leadingtothelossoffunction
ofthegene.So,knockingoutofageneiseasilyachievableusingconventionalCRISPR-Cas
technique.Furthermore,withthehelpofanarrayofgRNA,thesystemcanbeconveniently
used to target multiple genes at a time, a system is known as multiplex genome editing.
HDRmethodisexploitedforpreciseeditingwiththedeliveryofadonortemplatecontain-
ingthedesiredchanges,butitisinherentlylowinefficiencyinhighereukaryotes.
An efficient and convenient genetic transformation protocol is a prerequisite for the
success in genome editing by the CRISPR technology. There is no doubt that the rapid
development of the CRISPR-Cas tools has tremendously assisted biologists to adopt the
system for their specific purpose. However, the availability of a huge number of tools may
sometimesbeconfusingforanewusertochoosethemostsuitableforhis/herpurpose.We
hope that the ‘CRISPR-Cas Methods’ book would assist anybody who is new in this field
andneedadirectiontoadoptthistechnologyforaparticularorganism.
vii
viii Preface
This book details various CRISPR-Cas technical protocols for 11 different organisms
startingfromfungi,algae,planttohumancelllines.Asalreadymentioned,HDR-mediated
gene targeting in plant system is extremely difficult. In the very first chapter, to give an
overviewtothereaders,Mollaandco-workerssuccinctlydescribedtheprinciplesofdiffer-
ent CRISPR-Cas-derived technologies for basic biology, agriculture, and medicine. In the
Chapter2,HolgerPuchtaandcolleaguesdescribeanefficientprotocolforHDR-mediated
genetargetinginArabidopsisusinginplantamethodoftransformation.ThisStaphylococcus
aureusCas9(SaCas9)-mediated“inplantagenetargeting”(ipGT)approachinA.thaliana
should enable stable heritable genome modifications, including integration of a tag or cis-
element to a desired locus, or precise sequence modifications such as amino acid
substitutions.
Rice is core to the nutrition and livelihood of more than half of the world population
andplaysasignificantroleinworldfoodsecurity.InChapter3,LudwigandSlamet-Loedin
fromInternationalRiceResearchInstitute(IRRI)describeadetailedstepwiseprotocolfor
sgRNA design, cloning and assembly of CRISPR constructs, and analysis of mutants for
successful gene knockout. Base editing is a recently developed CRISPR-Cas-mediated
genome editing technique, which enables precise point mutation in the genome without
the need for a donor template. It enhances the efficiency of precise editing than that of
HDR. In Chapter 4, Molla and Yang described the step-by-step procedure of multiplex
adeninebaseeditingexperimentsinrice.Thisprotocolchapterwouldbeusefulforsimulta-
neousCRISPR-Cas-mediatedsinglebaseeditinginriceandothercropplantsatmorethan
onelocus.
DeborahPetrickelaboratelydescribedadetailedprotocolofhowtouseCRISPR-Cas9
editing tools to generate knockout mutant of the model grass species Brachypodium dis-
tachyoninChapter5.Availabilityofwholegenomesequence,brieflifecycle,simplegrowth
requirement, and its close relationship with most of the cereal crops and biofuel crops
switchgrass, make Brachypodium as a valuable model organism. Deb’s protocol would
significantlyexpeditebasicresearch,especiallyfor functionalgenomics.
Oomycete phytopathogens under the genus Phytophthora are destructive to many
economicallyimportantplantspecies.Molecular mechanismsunderlyingthepathogenicity
and broad host range of these fungi are poorly understood. CRISPR-mediated genome
editinghasbeenfoundasaneffectivetoolforoomycetefunctionalgenomicsforaccelerated
dissectionofgenefunctions.InChapter6,Tianandco-workersdescribedanovelprotocol
forgeneratingP.palmivoramutantsviaAgrobacterium-mediatedtransformation.
Chapter 7 by Cheng Dai and colleagues describes an efficient method for visual
screeningofmutants.Thisfluorescencescreeningmethodisvalidatedinseveraldicotspecies
like Arabidopsis, Brassica napus, strawberry, and soybean. The vector developed in their
studycouldbeappliedinmanyotherdicotplantspecies.
Similarly, Gasparis and Przyborowski in Chapter 8 and Adhikary and colleagues in
Chapter8outlinedprotocolsforapplyingthetechniquesinBarleyandFlax,respectively.
Twodetailedprotocolsforcarryingoutgeneknockoutexperimentsinhumancelllines
aredescribed.InChapter10,Uddinandco-workersdescribedacomprehensiveprotocolfor
gene deletion (knockout) and tagging (knockin) in mammalian cells, while in Chapter 11,
Semiech et al. described a protocol of cloning-free (DNA-free) CRISPR-Cas9-mediated
geneknockoutinhumanlivercellline.
Recently, DNA-free genome editing with direct delivery of pre-assembled CRISPR-
Cas9 ribonucleoprotein complex was successfully used to achieve targeted mutagenesis.
Genome-editing using DNA-free systems could circumvent restrictive GMO regulations,
thuspavingthewayfor widespreaduseofthisinnovativetechnologyintraitdiscoveryand
Preface ix
cropimprovement.Wheat(TriticumaestivumL.)isoneofthemostimportantcerealfood
cropsin theworld. This allohexaploid(AABBDD,2n ¼ 6x ¼ 42)isrecalcitrant togenetic
modification (GM). Chapter 12 by Bilchak and colleagues described the use of cell
penetrating peptides to deliver Cas9 ribonuclear protein complex to wheat microspore.
ThechapteralsodetailedontranscriptionalgeneregulationbyusingdCas9-VP64.Onthe
other hand, Bhowmik and Islam described a novel method for CRISPR-mediated wheat
geneeditingandtraitimprovementinChapter13.Theyprovidedastep-by-stepprocedure
thatbeginwithgRNAdesigningfollowedbyassemblingCRISPRconstructsorribonucleo-
protein complex and in vitro and in vivo gRNA validation using wheat mesophyll proto-
plasts, transformation and regeneration, mutation detection, and phenotyping for the
desiredtraits.
In Chapter 14, Tripathi et al. described the procedure for generating genome-edited
bananabyAgrobacterium-mediateddeliveryofthereagentstoembryogeniccellsuspension.
The Chlamydomonas reinhardtii is a microalgal model organism, for which a suite of
molecularandgenetictechniquesareavailable.InChapter15,FerencziandMolnardemon-
stratedtheuseofCRISPR-Cpf1inconjunctionwithsingle-strandedDNA(ssODN)repair
templatestoachievenucleargeneeditingefficienciesashighas30%.Itproducededitswith
predictableoutcomesinatransgene-andselectionmarker-freemanner.
Although there are many recent reports of successful CRISPR-Cas9-mediated gene
editing, the experimental tools required to implement this powerful technology is yet to
be embraced by most of the biological science laboratories as routine protocols. There are
several factors which affect the success and efficiency of CRISPR-mediated gene editing;
efficient gRNA designing and assembling multiple gRNA cassettes, selection of suitable
nuclease and base editors, efficient delivery of Cas9 and gRNA vectors, selection and
regeneration of edited organisms, efficient detection of the gene editing event, and so
on. The frequency of gene editing will depend on whether or not these conditions are
optimal. This volume of CRISPR methods and protocols is aimed to provide the funda-
mentalsofCRISPR-CassystemandtheadvancesintheprotocolsofCRISPR-Casgenome
editing for efficient genome editing. Contributors to this unique volume described their
reproducible experimental methods and protocols in 15 chapters and suggested many
troubleshooting ideas to avoid common CRISPR mistakes. Thus, this book will serve as a
laboratory manual providing readers a holistic view of CRISPR methodologies and their
practicalapplicationfor modifyingcropplants,microorganisms,andanimals.
This book is the outcome of a cooperative effort from all the editors and contributors
representingmanydifferentcountries.Theeditorsgratefullyacknowledgetheauthorswho
contributed to this book of CRISPR-Cas methods. Prof. Sophien Kamoun, FRS, The
Sainsbury Laboratory, Norwich, UK, deserves our sincere thanks for kindly writing the
Foreword of the book. Two coordinators from the Springer Jayashree Dhakshnamoorthy
andSanjanaSundaramalsodeservecreditsfortheirexcellentcoordinationwithauthorsand
editors.Ourthanksarealsoduetoothereditorialstafffortheirprecioushelpinformatting
andincorporatingeditorialchangesinthemanuscripts.Webelieveresearcherswhoworkor
would work on gene editing through CRISPR technology will find this volume as an
essentialguidebookforimplementingtheir researchprojects.
Gazipur,Bangladesh M.TofazzalIslam
Saskatoon,SK,Canada PankajK.Bhowmik
Cuttack,Odisha,India KutubuddinA.Molla
Contents
Foreword.................................................................... v
Preface ..................................................................... vii
Contributors................................................................. xiii
1 WideHorizonsofCRISPR-Cas-DerivedTechnologies
forBasicBiology,Agriculture,andMedicine ............................... 1
KutubuddinA.Molla,SubhasisKarmakar,andM.TofazzalIslam
2 EfficientHomologousRecombination-MediatedinPlanta
GeneTargetingbyEgg-Cell-SpecificExpressionofStaphylococcus
aureusCas9fromArabidopsis ............................................ 25
FelixWolter,Teng-KueiHuang,andHolgerPuchta
3 RiceGeneKnockoutorDownregulationthroughCRISPR-Cas9............. 35
YvonneLudwigandInezH.Slamet-Loedin
4 CRISPR-Cas-MediatedSingleBaseEditingatMorethan
OneLocusinRiceGenome.............................................. 51
KutubuddinA.MollaandYinongYang
5 CRISPR-Cas9-MediatedGenomeEditingoftheModel
GrassSpeciesBrachypodiumdistachyon .................................... 63
DeborahPetrik
6 CRISPR-Cas9-MediatedGeneEditingofthePlant
PathogenicOomycetePhytophthorapalmivora.............................. 87
MiaoyingTian,NatashaNavet,andDongliangWu
7 AnEffectiveCRISPR-Cas9TechnologyforEfficiently
IsolatingTransgene-FreeMutantsinArabidopsis,Brassica
napus,Strawberry,andSoybean .......................................... 99
ChengDai,HongYang,TingTang,ChaozhiMa,
andChunyingKang
8 AnOptimizedRNA-GuidedCas9SystemforEfficientSimplex
andMultiplexGenomeEditinginBarley(HordeumvulgareL.) .............. 117
SebastianGasparisandMateuszPrzyborowski
9 GenomeEditingofMammalianCellsUsingCRISPR-Cas:From
InSilicoDesigningtoIn-CultureValidation ............................... 143
BorhanUddin,PatrickPartscht,andTaslimaNahar
10 Cloning-Free(DNA-Free)CRISPR-Cas9-MediatedGene
EditinginHumanLiverCellLineandItsDetection........................ 163
MagdalenaS´miech,PawełLeszczyn´ski,EffiHaque,
andHiroakiTaniguchi
11 AProceduretoDesignGuideRNA,AssembleFragments,
andDetectMutationforGenomeEditinginFlax........................... 173
IsadoraLouiseAlvesdaCostaRibeiroQuintans
xi