APPLICATION NOTE GeneArt Genome Editing

APPLICATION NOTEGeneArt genome editingUsing Sanger sequencing to facilitate CRISPRand TALEN-mediated genome editing workflowsIn this application note, we show: Sanger sequencing by capillary electrophoresis can beused to determine the efficiency of genome editing inprimary transformed cultures Sanger sequencing is an efficient method to confirmsuccessful genome edits in transformed cultures, as wellas screen secondary clones for successful editing events Applied Biosystems Minor Variant Finder Software canbe used to determine the frequency of SNP changes inclones isolated from secondary culturesIntroductionEver since the double-helical structure of DNA waselucidated, researchers have developed techniques formanipulating DNA sequences. However, directing precisesequence changes at user-defined sites has remained adifficult and tedious challenge. Limited successes havebeen achieved with oligonucleotides, small molecules, orself-splicing introns, but the development of site-directedzinc finger nucleases (ZFNs) and TAL effector nucleases(TALENs) have facilitated sequence-specific manipulations.Nevertheless, difficulties of protein design, synthesis,and validation have slowed adoption of these engineerednucleases for routine use. The most recent gene editingtechnology, the CRISPR-Cas9 system, largely overcomesthese difficulties [1]. In fact, the CRISPR-Cas9 system hasproven to be so easy and inexpensive that one investigatorhas stated that it has brought about the “democratizationof gene targeting” [2]. Thus, the CRISPR-Cas9 system ispoised to transform genome editing.CRISPR-Cas9 technology is derived from a bacterialadaptive immune system. It is a two-component systemthat depends on an enzyme (Cas9) to cleave doublestranded DNA, and a guide RNA (gRNA) that directs theenzyme to the correct location in the genome. If a repairtemplate is not provided, the break produced by theenzyme is repaired by an error-prone nonhomologous endjoining (NHEJ) process. This results in a heterogeneouspopulation of cells with different insertions or deletions(indels) around the user-defined break. This process canbe exploited to generate cell lines with a specific geneknocked out. Alternatively, if a repair sequence is provided,the sequence around the break can be repaired using therepair sequence as a template. By selecting the sequencefor the repair template, precise sequence changes can beintroduced at a user-defined locus within the genome.However, to obtain a clonal population with a homologousgenome edit, several clones from the primary transformedpool of cells need to be screened. This necessitatestwo rounds of screening. First, a primary screen mustbe performed to determine the relative fraction of cellscontaining an edit. Knowing the efficiency of the edit willdetermine the number of single-cell clones that will needto be isolated for expansion. Next, a secondary screenmust be performed to identify the clones derived from asingle cell that have the desired edit. Sanger sequencingby capillary electrophoresis (CE) can provide informationat both screening stages. Sanger sequencing has beenthe gold standard for sequence determination for severalyears due to its simple, cost-effective workflow anduncomplicated data analysis. The data produced by CEallows unambiguous identification of sequence changes aswell as detection of mixed single nucleotide polymorphisms(SNPs) in a population. For these reasons, Sangersequencing by CE can be a valuable part of any genomeediting workflow.

Workflow overviewThermo Fisher Scientific hasintegrated all the tools necessary forgenome editing and downstreamanalysis (Figure 1). The Invitrogen GeneArt design tool facilitatesthe design and ordering of targetspecific gRNAs for CRISPR-mediatedgenome editing or TALs for TALENmediated genome editing. Invitrogen transfection reagents offer severaloptions for delivery of genomeediting tools into eukaryotic cells.In addition, Invitrogen TOPO TAcloning vectors and competent cellsfacilitate the sequence analysis ofprimary transformants. Gibco mediais available for growing the primarytransformants and secondary culturesfollowing clonal expansion. Finally,Applied Biosystems sequencinginstruments and reagents enable thedetermination of specific genomicediting events. In this applicationnote, we demonstrate how thisworkflow comes together to generateand identify mutations in the humanhypoxanthine phosphoribosyltransferase (HPRT) gene.A brief overview of the steps usedto generate and analyze a primaryculture with HPRT mutations isshown in Figure 2. The target-specificCRISPR RNA (crRNA) sequencewithin the gRNA was designed to aHPRT-specific locus. The gRNA wassynthesized via in vitro transcriptionusing the Invitrogen GeneArt Precision gRNA Synthesis Kit.Following synthesis and purification,gRNA was cotransfected withCas9 mRNA into 293FT cellsusing Invitrogen Lipofectamine MessengerMAX TransfectionReagent. The cells were harvested78 hours posttransfection. The celllysates were then used along withprimers flanking the HPRT targetto generate PCR amplicons noDesign andsynthesizegRNATransfectcellsDeterminefrequency ofsuccessfuleditsEstablishsingle-cellclonesScreen clonesfor editCharacterizesuccessfuleditsGeneArtCRISPRSearch andDesign Tool,GeneArtCRISPRreagentsGibco media,InvitrogentransfectionreagentsTOPO cloningkits, AppliedBiosystemssequencinginstrumentsand reagentsGibco mediaAppliedBiosystemssequencinginstrumentsand reagents,Minor VariantFinder or TIDEsoftwareAppropriateassay foryour systemFigure 1. Overall workflow for CRISPR genome editing. Thermo Fisher Scientific provides thetools, reagents, and expertise required for success at each step of the workflow.1.2. CRISPR-gRNAcomplex3. 4.5.Figure 2. Steps for determining the efficiency of an edit using TOPO cloning and Sangersequencing by CE. 1. Transfect gRNA and Cas9 mRNA into cells. 2. Incubate cells to allowprocessing of genomic change. 3. Purify genomic DNA from the cell culture, PCR-amplify theengineered locus from the heterogeneous culture, and clone PCR fragments into TOPO vector.4. Isolate plasmids from single colonies and PCR-amplify the insert. 5. Sequence the insert. Theefficiency of the edit is the ratio of the number of inserts with an engineered change to the totalnumber of inserts sequenced. Higher efficiency will likely result in fewer secondary clones that needto be screened to identify specific cells with the change.greater than 600 bp in length. The PCR products were then subcloned usingthe Invitrogen Zero Blunt TOPO PCR Cloning Kit and transformed intoInvitrogen TOP10 E. coli cells. Ninety-six bacterial colonies were picked pertransformed pool of gene-edited cells and processed for DNA isolation using theInvitrogen PureLink 96 Plasmid Purification System and subjected to Sangersequencing. The resulting sequencing data was then analyzed to measure thepercent of PCR products containing accurately edited sequence and to selectwhich clonal isolates to maintain. Alternatively, although it was not performedfor this study, the PCR product could be sequenced directly, without subcloninginto TOPO cells.

Demonstration of sequencingresults in the primary screenIn any genome editing experiment,the nuclease cleavage and repairprocess is not completely efficient oraccurate. Therefore, before moving onto clonal isolation of engineered cells,the fraction of cells containing an editshould be determined. One way todo this is to PCR-amplify the regionedited from primary transformantcultures and subclone into a plasmid.By sequencing a large number ofplasmids, the fraction containing anedit can be determined. This alsogives a first glance into the overallgene knockout or editing efficienciesand type of indel changes that mighthave occurred.After transfecting 293FT cellswith gRNA and Cas9 mRNA, wesubcloned the locus from primarytransformants and sequenced 96clones. Of those 96 clones, 84 alignedwith the target sequence. Only 12clones had no editing event in theamplified region. Seventy-two cloneshad at least one sequence deviationfrom the wild-type sequence, for anoverall efficiency of about 86%. Thisgave us a good idea of how manysecondary single-cell clones neededto be screened to find a desired pureknockout clone. Interestingly, someof the TOPO clones had a mixedsequence (Figure 3). This could be dueto either the bacterial colony havingtwo distinct plasmids, or the DNA notbeing derived from a single colony.Nevertheless, it is clear that an editingevent is present. Note that sequence isuniform up to the red arrow. After that,each position consists of two peaks,indicating two different sequencesare present. It is not easy to separatethe sequences at this level; however,it is clear that CE sequencing canshow that an edit occurs at thecorrect location even if downstreamsequences can’t be read accurately.Figure 3. Example CE trace of a mixed clone, containing two different edited sequences.Notice that the sequence is uniform up to the red arrow, after which there are two differentsequences present in approximately equal amounts. Because one sequence contains a deletion, it isout of register with the other sequence and can’t be easily read.Figure 4. Sequences within the HPRT locus produced by NHEJ of DNA following cleavage bythe CRISPR-Cas9 system. Genomic editing events can produce a variety of sequence changes,especially in the absence of a repair template. Each line of sequence shown is derived from adifferent TOPO clone and aligned to show differences. The entire guide RNA sequence used isshown at the top; the boxed sequence emphasizes the regions shown below. The normal HPRTlocus is labeled in dark blue on the left. Yellow boxes with red font are nucleotides that are identical towild-type HPRT (unchanged); blue and white boxes illustrate nucleotide differences.Analysis of sequences present in many different colonies revealed the spectrumof changes introduced by the editing complex (Figure 4). Each sequence shownwas from a different TOPO clone and represented a different molecule in theprimary transformant culture. Deletions and insertions are apparent around thegRNA site and are not confined to a specific base. Since the editing complexcan introduce a wide variety of changes, a collection of clones derived from theprimary transformant culture should be sequenced to profile and predict whatedits may be expected in the secondary screen.Demonstration of sequencing results in the secondary screenAfter generating a primary transformed culture, and while that culture wasbeing characterized, single cells from the heterogeneous primary culturewere obtained by limiting dilution. Clones were grown for 14 days, and lysedas described in the Invitrogen GeneArt Genomic Cleavage Detection Kitmanual. Sequences of the locus around the putative edit were PCR-amplifiedusing target-specific primers (forward: 5 -GTGTTAATTTCAAACATCAGCAGC-3 ,reverse: 5 -GTCTTCTTGTTTATGGCCTCC-3 ). The resulting PCR productswere subjected to Sanger sequencing by CE and analyzed using AppliedBiosystems Sequence Scanner Software v2.