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Aftab Ahmad Sultan Habibullah Khan Zulqurnain Khan Editors The CRISPR/ Cas Tool Kit for Genome Editing The CRISPR/Cas Tool Kit for Genome Editing (cid:129) (cid:129) Aftab Ahmad Sultan Habibullah Khan Zulqurnain Khan Editors The CRISPR/Cas Tool Kit for Genome Editing Editors AftabAhmad SultanHabibullahKhan DepartmentofBiochemistry/US-Pakistan CenterofAgriculturalBiochemistryand CenterforAdvancedStudiesin Biotechnology(CABB)/US-PakistanCenterfor AgricultureandFoodSecurity AdvancedStudiesinAgricultureandFood UniversityofAgriculture Security Faisalabad,Pakistan UniversityofAgriculture Faisalabad,Pakistan ZulqurnainKhan InstituteofPlantBreedingand Biotechnology MuhammadNawazSharifUniversityof Agriculture Multan,Pakistan ISBN978-981-16-6304-8 ISBN978-981-16-6305-5 (eBook) https://doi.org/10.1007/978-981-16-6305-5 #SpringerNatureSingaporePteLtd.2022 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthe materialisconcerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation, broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionorinformation storageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilarmethodology nowknownorhereafterdeveloped. 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The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore Foreword On the invitation of Dr. Aftab Ahmad, Dr. Sultan Habibullah Khan, and Dr. Zulqurnain, I am pleased to write introductory statements about The CRISPR/ Cas Tool Kit for Genome Editing, a very informative book about developments in CRISPR/Casanditsapplications. The emergence of CRISPR/Cas and its re-engineering into a potent genome editing tool has revolutionized the life sciences with a broad range of applications inbasicandappliedresearch.Ithasbroughtmuchexcitementandhopeinmedicine, agriculture, and food security with unprecedented control over the redesign of genomes, which has been recognized through the Nobel Prize for CRISPR in 2020. Derived from bacterial adaptive immune system against foreign DNA, CRISPR is a simple, efficient, and robust platform for genome editing in bacteria, plants, and animals. CRISPR has become a most favored method to create animal models for studying molecular mechanisms, fighting viral, bacterial, and fungal diseases in plants, eradicating vector-borne diseases such as Zika, dengue, and malaria, repairing disease-causing mutations, and many others. The CRISPR tool kit is continuously growing with the latest addition of Cas13 for transcriptional control, Cas14 for ssDNA, and EvolvR for rewriting genetic code. No doubt, the CRISPR-Cas system has transformed biological research with new tools, latest methods, and breakthrough discoveries of science. Although CRISPR has empowered scientists with unprecedented tools of precise genomic editing but CRISPR is at its developing stage and we can expect that best in CRISPR is yet tocome. This comprehensive book aims to provide the reader with basic and up-to-date knowledge about CRISPR/Cas-mediated genome editing and different techniques basedonthiscutting-edgetechnology.Itcontainstenchaptersaboutvariousaspects ofCRISPR/Casfromintroductiontochallengesandprospectsofthisrevolutionary toolbox. It also contains a chapter discussing directed evolution focusing on rewritingofgeneticcodeusingCRISPR/Castoevolvefunctionsofproteinsbeyond natural evolution. In addition, one chapter highlights the importance of CRISPR/ Cas13 with its detailed application in RNA world such as RNA editing, RNA imaging,anddiagnostics.Moreover,achapterprovidesinsightsintotheapplications of CRISPR/Cas beyond genome editing, which are exceeding genome editing vv vi Foreword aspects.Theeditorsarealsoauthorsofmostofthechapters,whohaveanestablished expertiseongenomeeditingwithCRISPR/Casinplants. IamhappytorecognizethisvaluableworkofDr.A.Ahmad,Dr.S.H.Khan,and Dr. Z. Khan for academic and scientific readers with leading science publisher Springer Nature. I believe this volume provides a clear, informative, and an easy- to-understandtextonCRISPR-mediatedgenomeediting.Iamhappytosaythatthis book will be useful particularly for students, researchers, and scientists to gain insightaboutCRISPR/Cas. StateKeyLaboratoryofPlantCelland CaixiaGao ChromosomeEngineering,InstituteofGenetics andDevelopmentalBiology,CAS Beijing,China Preface CRISPR/Cas-basedgenomeeditinghasbecomeanewfaceofmolecularbiologyas it has been adopted in almost every filed of biotechnology from agriculture to medicine. CRISPR/Cas is continuously evolving with new techniques and new Cas proteins with diverse functions. In the past few years, there have been rapid advancementsinonlinetools,methods,protocols,andapplicationsofCRISPR/Cas technology.Fine-tuningandcontinuousdevelopmentsinCRISPRtechnologyhave allowed scientists to change single base pair with more precision and accuracy. In addition, CRISPR-based directed evolution has enabled us to rewrite genetic code and discover functions of proteins beyond natural evolution. This contributed volume,TheCRISPR/CasToolKitforGenomeEditing,providesacomprehensive overview ofCRISPR-based tools from theirdiscovery, development,anddiversity to their applications in various organisms. Starting with an introductory chapter about CRISPR/Cas, the book aims to bring details about CRISPR applications in genome editing, which makes this volume equally helpful for readers of various levels. The chapters of this book not only summarize historical background and classification of CRISPR/Cas but also discuss a variety of CRISPR/Cas systems such as Cas9, Cas12, Cas13, CasX and their applications including multiplex genomeediting,baseediting,primer editing,RNA editing, and directedevolution. In addition, contents of this book also discuss design tools for determining on-targeting and off-targeting, and different delivery methods for efficient genome editing. This book has been designed for students, researchers, and professionals inter- ested in CRISPR/Cas and want to learn more about it. The book brings together differenttoolsavailableinCRISPR/Castechnologyforgenomeeditingapplications in bacteria, animals, plants, and model organisms. Re-engineering CRISPR/Cas systemanditsuseforre-writinggeneticcodesprovideanadditionalaspecttogain more attention of the advanced level researchers in the field of genome editing. Trends in the CRISPR technology, its applications and regulation are chalked out alongwithaddressingseveralissuessuchasoff-targeting,regulatoryconcerns,and biosafety. The work presented here may be a valuable resource for developing an academiccourseonCRISPR-basedgenomeediting.Indeed,thisnicelywrittenand well-presentedworkisavaluableadditiontothefieldofgenomeediting,whichwill help the scientific community to learn, understand, and apply the marvelous vii viii Preface technologyforthewelfareofhumankind.Havingequalimportanceforundergradu- ate and postgraduate students to researchers and scientists, the book will also be foundusefulforpolicymakersandindustrialstakeholders. Faisalabad,Pakistan AftabAhmad Faisalabad,Pakistan SultanHabibullahKhan Multan,Pakistan ZulqurnainKhan Contents 1 AnIntroductiontoGenomeEditingTechniques. . . . . . . . . . . . . . . 1 NaylaMunawarandAftabAhmad 2 HistoryandClassificationofCRISPR/CasSystem. . . . . . . . . . . . . 29 ZulqurnainKhan,ZulfiqarAli,AsifAliKhan,TahminaSattar, AliZeshan,TahiraSaboor,andBarkhaBinyamin 3 BioinformaticToolsinCRISPR/CasPlatform. . . . . . . . . . . . . . . . . 53 AftabAhmad,SidraAshraf,HumeraNazMajeed,SabinAslam, MuhammadAamirAslam,MuhammadSalmanMubarik, andNaylaMunawar 4 DeliveryMethodsforCRISPR/CasReagents. . . . . . . . . . . . . . . . . 113 SidraAshraf,NaylaMunawar,MuhammadKashifZahoor, AmerJamil,MasoomaHammad,AbdulGhaffar,andAftabAhmad 5 ReengineeringoftheCRISPR/CasSystem. . . . . . . . . . . . . . . . . . . 149 ZulqurnainKhan,TahminaSattar,MariaSiddique,ZulfiqarAli, AsifAliKhan,SyedOvaisAftab,MuhammadZubairGhouri, QaisarSultan,NaumanGulzar,andFarooqAhmad 6 BeyondGenomeEditing:CRISPRApproaches. . . . . .. . . . . . . . .. 187 QaisarSultan,SidraAshraf,AhmadMunir,SultanHabibullahKhan, NaylaMunawar,KamelA.Abd-Elsalam,andAftabAhmad 7 RNAEditingwithCRISPR/Cas13. . . . . . . . . .. . . . . . . . . . . . . .. . 219 SidraAshraf,MuhammadZubairGhouri,MuhammadAsifJaved, HassanZafar,HazratAli,SameerH.Qari,andAftabAhmad 8 CRISPRforRewritingGeneticCode. . . . . . . . . . . . . . . . . . . . . . . 255 ZulqurnainKhan,AsimRazzaq,AyeshaMuneer,MariaSiddique, UmarIqbal,MuhammadAsifRaza,andTahminaSattar iixx x Contents 9 ApplicationsofCRISPR/CasSysteminPlants. . . . . . . . . . . . . . . . 285 SultanHabibullahKhan,HooranTariq,IqraFarooq,HiraTasleeem, MuhammadZubairGhouri,MuhammadSalmanMubarik, andZulqurnainKhan 10 ChallengesandFutureProspectsofCRISPRTechnology. . . . . . . . 311 ZulqurnainKhan,SultanHabibullahKhan,andAftabAhmad