CRISPRengineering(DNA,RNA,or DNA/RNA)ServiceForm

Noguaranteeismadeforthenumberoftransgenicmiceproducedsincethisdependsona numberoffactors including:

1) ThequalityofDNAs/RNAsprovidedbytheinvestigator(ifprovidedbytheinvestigator)

2) TheabilityoftheguidetotargettheCas9tocutinvivo

3) Thechromatinstructure/accessibilityoftheDNAsequencetobetargeted(highlyvariableand unpredictable)

Please check the box next to the indicated responsibility as to whether the GTTR (Gene Targeting and Transgenic Resource) is responsible, or the Investigator lab is responsible:

Task / Responsibility of GTTR Core / Responsibility of Investigator Lab
ToprovideDNA vectorsand/orRNAsas specified(seetheformforspecificquantities,volumesand concentrations),tobedeterminedby mutualagreementwithstaff.
Toprovidea descriptionoftheDNAvectorsand/orRNAsinvolved,anda briefdescriptionoftheexperiment.
To providesequenceofCRISPRRNAsused,donorDNAmoleculestobeusedforHDR,ifany(e.g.DNAoligosor fragments),andthesourceofCasforCRISPRinjections.(DescribeoriginofCRISPRbackboneandCasgene beingused.)
ToassurethattheDNAand/orRNAfragment(s)willnotproduceanycontagiousconditionthatmaybe harmfultoanimalsortohumansandthattheydonotexceedcontainmentrequirementsgreaterthanthose specifiedbyNIHBL-2standards.
ToinformthenumberoftransgenicmiceobtainedaftertailDNAanalysisbySouthernorPCRwithin1monthofbeingnotifiedthattailsamplesarereadyfor screening.

Recommendednumberofinjectiondaystorequest:Becausethenumberofembryosavailableforinjectioneach daywillvary,weguaranteeatleast150-300embryoswillbeinjectedper scheduledproject.Theactualnumber injectedmaybemorethanthis,butwillnotbeless orthestrainofthemousechosen.

CRISPRProject:RequestInformation

Date:

Investigator:

Institute:

Address:

E-Mail:

Phone Number:

IACUC/animalwelfareprotocolnumber: _

DescriptiveNamefor thisCRISPRProject

Pleaseprovidea briefbutclearlyidentifiablename.(Willappearon cagecardaswrittenhere):

Briefdescriptionofdesiredoutcomeofprojecti.e.whatisthemouseyouwanttomake?

CRISPR-mediatedcleavagecanresultinmutagenesisofbothcopiesofthetargetgene. Couldinactivationof thetargetgene(s)causelethalityor abnormalphenotypes,eitherintheheterozygousor homozygousstate?If sopleaseexplain.

ForCRISPRproject: Pleasecompletethissection.

TargetedGene(s)MGIID:

TargetedGene(s)Name:

TargetedGene(s)Symbol:

PleaselistthenamesandprotospacersequencesofanyCRISPRguideRNAstobeinjected (alternatively, if the GTTR does the design and production, we will fill out and return back to you):

*FortheembryonicexpressionoftheCRISPRandCas9components,whichtypesofnucleicacidswillbeinjected:

eitherDNAplasmids,orinvitrotranscribedRNAs? (Checkone)DNA

RNA _ Both_

IfyouansweredDNAtothepreviousquestionmarkedby *, completethe followingsection. IfyoumarkedRNA, skipthissection.

DescribetheDNA vector(s)used:

Vectorsize:

MethodofvectorDNApurification:

IfyouansweredRNAtoquestionmarkedby *, completethefollowingsection.IfyoumarkedDNA,skipthis section.

Cas9RNA:Providedbyoutsidevendor?Yes _No _ If Yes,listthevendor:

IfNo,pleaselistthesourcetemplateplasmid,synthesisandpurificationmethods:

Cas9Template:

Cas9SynthesisandPurificationmethods:

Finalconcentrationsofoligo/guide/nucleasewillbeasfollows: EachInjectedoligowillbe100ng/ul;

EachGuideRNAwillbe50ng/uland

Cas9 willbe20ng/ul.

Pleaseplanaccordinglyastheyneedtoberesuspendedtogetherata finalinjectionvolumeof50uL/day(atleast).

Deviationsfromtheseconcentrations,ifrequested,shouldbeexplainedbelow:

ForallCRISPR projects: DoesthisinjectionalsorequireinjectionofadditionalDNAcomponents,suchasdonor molecules(oligosorDNA fragments)thatareintendedto edit,recombinewith,orinsertintotheCRISPRgenomic targetsite(s),asviathehomology-dependentrepairpathway?

Yes _No _

Iftheanswertothelastquestionisyes,listanddescribeanyDNAoligosorfragmentsusedincombinationwith

CRISPRRNAsforgeneediting(providetheseDNA sequencesina separatedocument)(alternatively, if the GTTR does the design and production, we will fill out and return back to you):

DoyouhaveanypriorevidencethattheexactsameCRISPRtarget(s)cangeneratemutationswithefficiency?(E.g. priorinjectionortransfectiondatausingthesameCRISPR).If sopleasedescribethe experimentandrecorded efficiency:

PleasedescribethemethodsthatwillbeusedtoscreenandverifyanyCRISPR-generatedmutations/genome editinginthefounderanimals(e.g.Surveyorassays,directsequencing,orother)(alternatively, if the GTTR does the design and production, we will fill out and return back to you):

Strainofmicedesiredforinjections: _(B10xC3Hhybrid;C57BL/6J;other?)

Providea briefdescriptionoftheintendeduseoftheengineeredmicetobegenerated:

FOR CRISPR PROJECT REQUESTS,Pleaseattach tothisform(alternatively, if the GTTR does the design and production, we will fill out and return back to you):

1. SequencesofguideRNAsand any DNA oligos/donorfragmentsbeing injected

2. Ifavailable,photosof gel analysisof RNAs.

3. ExampleofPCRdemonstrating successful amplification of thewild-typemutation targetfrom mousegenomic DNA.

CRISPR-CasDisclaimers:

•CRISPR-Cas-mediated “genome editing”is arapidlyemerging technology.Although thecorehas demonstrated successful invivotargetcleavageinmouseembryosin alimited numberof experiments, notalltargetsmayinducegenomeediting equallywell,andsotheGTTRcannotguarantee successful cleavage/editing. Itistheresponsibility oftheinvestigatortoconfirmwhethermutagenesis and/orgenome editing hasoccurred successfullyin theresultingmice.

•SgRNA-mediated cleavagehasbeen reportedto be pronetooff-targetmutagenesis. Theseevents havebeenobserved in someCRISPR-modifiedmice,although notall CRISPRguideRNAswill behighly pronetothisproblem. TheGTTRdoesnothold responsibilityforoff-targetmutations.

• DNAcleavageoftenoccursatthe1-cell stage,but mayoccur atthe2-cell stageor later. CRISPR- mediatedmutagenesiscanthereforegeneratemosaicanimalswith regardstotheproportion ofcells thatcarryspecificmutations. On the other hand,morethan2typesofmutationsatasingletargetsite can sometimesbeobserved in tissuesfromtheinjected mice.Thus,PCRdetection ofmutationsin livebornmicedoesnot guaranteegermlinetransmission ofparticularmutationsis efficient orpossible (although ittypicallyshould be).

•CRISPR-Cas-mediated mutagenesiscanaffectboth allelesin a1-cell embryo.Thus,increased lethality mayresultfromtargetingessential genes,reducing yield of livebornpupsand/orcorrectlytargeted

pups

• Micewill bereleased/shipped to theinvestigatoronlywhenpaymentfortheprojecthasbeenmade,

and when proper animal transportpaperworkiscompleted.

Investigator’ssignature:

◎Please submit the completed form to .

Beijing SBS Genetech Co. Ltd.

Fax: +86-10-82784290

Email: ,

Website: