Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs

Domain: biology
Published: 2026-01-15
Editorial outcome: minor_revision

Abstract
CRISPRnuclease-mediatedgeneknock-outislimitedbysuboptimalsgRNAs,
inaccessibletargetsites,andundesiredrepairoutcomes.Here,wepresenta
Cas12a-basedsysteminDrosophilathattargetseachgenewithfoursgRNAsto
overcometheselimitations.MultiplexedsgRNAsactthroughredundancyand
synergism,frequentlycreatingdeletionsbetweentargetsitesandincreasing
thefractionofloss-of-functionmutations.Weshowthatmultiplexedgene
targetingiswelltoleratedanddoesnotcausewidespreadproximityeffects.To
visualizeCRISPR-nucleaseactivityinlivinganimals,wedevelopedascreening
assayandusedittoassessCas12aactivityacross33%oftheDrosophilagenome
incombinationwithover2000sgRNAs.Thisrevealedremarkablyhighon-
target(>99%)andverylow(<1%)off-targetactivityofmultiplexedCas12a
sgRNAarrays.Quantitativeside-by-sidecomparisonswithcurrentCas9-based
systemstargetingover100genesinparalleldemonstratethatmultiplexed
Cas12agenetargetingachievessuperiorperformanceandrevealsphenotypes
missedbyestablishedmethods.Thesystemdescribedhereprovidesa
frameworkforreliablegeneknock-outinmulticellularsystems.

Introduction
CRISPR gene editing has significantly accelerated the functional
annotationofgenomes1–3.Despiteitstransformativeimpact,several
factors limit the efficiency of gene disruption through CRISPR
nucleases.Forexample,suboptimalactivityofsgRNAsorCasproteins
can impede the induction of DNA breaks and chromatin state and
geneticvariationcanrendertargetsitesinaccessible.Moreover,repair
ofDNAdoublestrandbreaks(DSBs)typicallyresultsinaspectrumof
mutations that includes undesired repair outcomes retaining gene
function,suchassmallin-frameinsertionsordeletions(indels).Asa
result, CRISPR editing in multicellular systems produces genetic
mosaics - populations of cells carrying different mutations or
maintaining wild-type sequences. This severely compromises the
sensitivityandrobustnessoffunctionalgenediscoverywithCRISPRin
animals,plantsandculturedcells,presentsahurdleforthedevelop-
mentofgeneticcontrolstrategiesfordiseasevectors,andlimitsits
potentialtherapeuticapplicationincellandgenetherapy.

Apromisingstrategytoovercometheselimitationsisthesimul-
taneoustargetingofgeneswithmultiplesgRNAs4–6.Here,additional
sgRNAs can compensate for those that fail to mediate the desired
outcome.However,forCas9,themostwidelyusedCRISPRnuclease,
creatingmultiplexedarraysofmorethantwosgRNAsatscaleremains
technicallychallenging.Thelargesizeandrepetitivenatureofsuch
arraysprecludesthemfrombeingsynthesizedbycommercialprovi-
dersandmanualassemblyistimeandcostintensive4,6.Mostcurrent
large-scaleCas9sgRNAlibrariesthusemployonlyoneortwosgRNAs
per gene7–12. In contrast, Cas12a nuclease is guided solely by small
crRNAs(hereaftersgRNAs)andcanautonomouslyprocesscompact
arraysintoindividualsgRNAs13.ThesepropertiesallowCas12asgRNA
arrays to be encoded on commercially available oligonucleotides,
substantially facilitating generation of expression plasmids at scale.
Thishasbeenharnessedformultiplexgeneeditingandtranscriptome
engineeringinvariouscontexts5,14–17,butwhetherhigher-ordermulti-
plexingofsgRNAstargetingthesamegeneenhancestheefficiencyof
genedisruptioninvivoremainsunclear.

A key challenge of CRISPR gene editing is the occurrence of
undesiredon-andoff-targetmutationsthatobscurethephenotypic
effects of disrupting the target gene. Large deletions, chromosome
arm truncations and chromothripsis can arise from double strand
breaksattheon-targetsiteandcompromiseunrelatedgeneslocated
incis18–22.Sucheventshavebeendemonstratedtooccurwithrelevant
frequenciesinmammaliancellsandsomeotherorganisms,buttheir
relevanceforgenetargetinginDrosophilahassofarnotbeeninves-
tigated.Inaddition,off-targetmutationscanarisewhenCasnucleases
cleave distant sites with imperfect homology to the sgRNA23,24.
Althoughcomputationaltoolscanpredictsomeoff-targetsites,their
reliabilityislimitedbypoorlyunderstoodsgRNA-specificeffectsand
sequencedifferencesbetweenreferenceandexperimentalgenomes.
However, methods forthe unbiased, experimental detection of off-
targeteffectsinthegenomeofmostanimalsarelimited.Methodsfor
thedirectdetectionofinducedDNAbreakshavebeendescribed,but
arenotavailableformostspeciesduetotheabsenceofspecialized
toolslikeantibodies binding specificDSBrepairproteins25,26.Whole
genomesequencingisbroadlyavailable,butitsapplicationforcom-
prehensiveoff-targetanalysisacrossmanysgRNAsislimitedbycost.
Moreover,sinceCRISPR-inducedmutationsandnaturalgeneticvar-
iationareoftenindistinguishable,inferringcausalrelationshipsabout
off-targeteffectsfromsequencingdataaloneremainsnon-trivial27,28.
Consequently, the off-target activity of CRISPR systems in many
organismsremainsunclear,asmanystudiesevaluateactivityatonlya
verysmallselectionoflociorwithasmallnumberofsgRNAsordonot
testforoff-targetcleavageatall.

Here, we describe a collection of optimized, inducible Cas12a
transgenesandquadruplesgRNAarraysfortargetedmutagenesisof
over 800 genes. Multiplexed sgRNAs provide redundancy and act
synergistically to frequently induce deletions between target sites,
generatingmutationsthataremorelikelytodisruptgenefunction.We
performed large-scale activity screening and benchmarking against
currentCas9-basedtechnologies,whichrevealedveryhighon-target
activitywithmorethan99%oflinesbeingactive,anddemonstrated
superiorknockoutperformanceacrossmanytargetgenesandtissues.
We also systematically investigated potential undesired effects
including toxicity, proximity effects, and off-target activity. These
analysesrevealedthatmultiplexedgenetargetingiswelltoleratedand
highly specific, with perturbations highly confined to the on-
targetlocus.

Results
SynergisticactionofquadrupleCas12asgRNAarrays
WesetouttoimplementandevaluatemultiplexedCas12agenetar-
getinginthetractableDrosophilamodelsystem.Theshortgeneration
timeof this organismenables comparisons across many conditions
and replicates and the ability to encode nucleases and sgRNAs on
single copy transgenes in defined genomic landing sites eliminates
variations in delivery efficiency or expression level as confounding
factorsofexperimentalresults.Wepreviouslyshowedthatavariantof
LachnospiraceaebacteriumCas12awithaD156Rmutation(hereafter
Cas12a+)functionswithhighefficiencyinDrosophilaandcanprocess
largesgRNAarrays29.Topavethewayforthecreationofsystematic
resources for multiplexed Cas12a+ gene targeting, we firstaimed to
determinetheoptimalnumberofsgRNAs.Wedevelopedareporter
assayusinggenomicinsertionsofanexogenoussequencecontaining
onetosixtargetsitesofaknown,highlyactivesgRNA(Fig.1a).This
representsacontrolledsetupdevoidofthetypicalvariationsinDNA
cutting activity when different sgRNAs are used. Gene editing with
ubiquitouslyexpressedCas12a+andsingleormultiplexedsgRNAtar-
getsitesgeneratedavarietyofeditingoutcomesinsomaticcellsand
thegermline.Editingatsingletargetsitesprimarilyproducedsmall
indels,whiletargetingmultiplesitesprogressivelydiversifiedthetar-
getlocusandinducedlargerdeletionsbetweensites.Thiswasevident
from the migration pattern of amplicons from the target locus in
somatic cells(Fig. 1b), whichprogressively transitioned toa ‘smear’
indicating heteroduplex DNA formation during PCR, and from
sequencing reads of individual germline-transmitted alleles, which
revealedfrequentdeletionsbetweentargetsites(Fig.1c).Weselected
adesignwithfoursgRNAspergeneforin-depthcharacterizationto
balance the benefits of multiplexing against potential drawbacks,
whichcouldincludeexcessiveDNAdamage,proximityeffects,andoff-
targetactivity.Thisdesignchoiceisfurthersupportedbyrecentevi-
dence that arrays consisting of more than 4 sgRNAs are no longer
optimallyprocessedbyCas12a5.

Next,weevaluatedtheeffectsofmultiplexedgenetargetingon
theintegrityofeightendogenousloci.Here,differentsgRNAsareused
to target independent sites within each gene, which is expected to
result in more diverse editing outcomes compared to the reporter
system described above. PCR amplicons from genes targeted with
quadruple sgRNA arraysand Cas12a+ showed heterogeneous migra-
tionpatternsduringagarosegelelectrophoresis(Fig.1d).Weobserved
a pronounced decrease in full-length amplicon abundance with a
concomitantincreaseinalternativeproductsforsixtargetgenes,an
effectthatwasweakerforonegeneandabsentforanother.Inparallel,
wetargetedthesamegeneswithlinesofourpreviouslydescribedCas9
sgRNAlibrary,whichusestwosgRNAspergene10.Usingthisdesign,
the reduction in full-length amplicons was substantially less pro-
nounced,beingstrongforone,butweakorabsentfortheremaining
seventargetgenes.

Together,theseresultssuggestthatCas12a+-mediatedtargeting
withquadruplesgRNAarraysproducesgreatertargetlocusdiversifi-
cationcomparedtodual-sgRNACas9editing.Notably,thiscompar-
ison contrasts the system described here with the dual-sgRNA
approachusedinmanypubliclyavailableDrosophilasgRNAlinesand
does not constitute a direct comparison of nuclease performance,
which would require matched sgRNA designs. Collectively, these
findings, together with published reports4,30,31, suggest that sgRNA
multiplexingcouldenhancegenedisruptionthroughtwomechanisms
(Fig. 1e). First, multiple sgRNAs provide functional redundancy,
allowingactiveguidestocompensateforinactiveonesorthosepro-
ducingsilentmutations.Second,multiplexedsgRNAscanactsyner-
gistically by generating simultaneous cuts that create deletions
betweentargetsites,which are morelikely to yieldloss-of-function
allelesthansmallindels.

GenerationofamultiplexedCas12asgRNAlibrary
To facilitate conditional gene knock-out in Drosophila with multi-
plexed sgRNAs and Cas12a+ we created a large, publicly available
toolbox of sgRNA and nuclease expressing fly strains. First, we
designed a library of quadruple sgRNA arrays using our previously
describedCRISPRLibraryDesigner32.sgRNAswereselectedaccording
toseveralcriteria,includinglackofpredictedoff-targetsites,targeting
ofcommonexons,optimizationoftargetsitepositionandspacing,
and target site-specific parameters (see methods). sgRNAs are
expressedfromthestrong,ubiquitousU6:3promoterandintegrated
atadefinedattPlandingsiteonthesecondchromosome(Fig.2a).The
abilitytoencodeallfoursgRNAsonasingleoligonucleotideallowed
forsynthesisandcloninginapooledformat,resultinginsubstantial
savings in cost and time compared to the construction of our pre-
viouslydescribedCas9dualsgRNAlibrary(seemethods)10.Sofar,we
have created Cas12a sgRNA lines targeting over 800 unique genes
(Supplementary Data 1). We refer to these lines as the HD12aCFD
sgRNAlibrary.Inparallel,wealsocreatedoptimizedCas12a+expres-
sion constructs (Fig. 2b). These feature a Gal4-dependent UAS pro-
moterfortissue-specificexpression,anupstreamopenreadingframe
to avoid excessive expression levels, and dual nuclear localization
signalsforefficienttranslocationintothenucleus(Fig.2b,andSup-
plementaryFig.1).Tofacilitatecombinationofthesetransgeneswith
different Gal4 drivers, we created independent insertions of the
Cas12a+-expressionplasmidsonallthreemajorchromosomes.Wealso
constructed an additional line in which Cas12a+ expression can be
controlledthroughexcisionofaFRT-flankedGFPcassette(Fig.2b,and
SupplementaryFig.1).

Lastly, we generated a reporter that can be used to visualize
CRISPR-Cas12a activity in vivo (Fig. 2c). Analogous to previously
describedreportersforCas9activity33,34,thisconsistsofaninhibitory
upstreamopenreadingframeinfrontofaGFPgene,whichcanbe
targeted by a specific Cas12a or Cas9 sgRNA. Mutagenesis of the
reporter leads to the expression of GFP, whichcanbevisualized in
livinganimalsorfixedtissues(Fig.2d).Duetohighactivityandthe
permanentnatureoftheinducedmutations,CRISPRmutagenesiscan
deviate from established Gal4 expression patterns (Fig. 2e-g)10. It is
thereforeadvisableto empiricallyestablishthe activity patternof a
newlygeneratedGal4UAS-Cas12a+line.

MultiplexedCas12agenetargetingofsingleginesiswell
tolerated
Before exploring further the knockout efficiency of multiplexed
Cas12a+genetargeting,weinvestigatedpotentialadverseeffectsof
usingseveralsgRNAstodisruptthesamegene.SinceDNADSBsare
detrimental to cell fitness, one concern is that inducing multiple
breakssimultaneouslycouldoverwhelmtheDNAdamageresponse,
leadingtoexcessivecelldeath35–37.Toassessthisrisk,wequantified
apoptosislevelsfollowinggenetargetingwithmultiplesgRNAsby
stainingcellsforactivatedeffectorcaspaseDCP-1.InductionofDSBs
in non-essential genes caused a moderate increase in apoptosis
compared to control tissue without DNA damage (Fig. 3a, b).
Importantly, the level of cell death was not significantly different
whethergenetargetingwasperformedwithCas9andtwosgRNAsor
Cas12a+ and four sgRNAs (Fig. 3a, b). This finding aligns with our
functional validation experiments (see below), where multiplexed
targeting of characterized genes using HD12aCFD yielded exclu-
sively target-specific phenotypes. We also assessed toxicity after
targeting genes present on multiple chromosomes as a result of
theiruseastransgenicmarkers(SupplementaryFig.2).Weobserved
a marked increase in apoptotic cells in these genotypes. This
was independent of the total number of sgRNA target sites, as a
genotype with only four target sites scattered across the genome
resultedinmorecelldeathcomparedtoeightsitesclusteredata
single locus (Fig. 3a, b, and Supplementary Fig. 2). These results
indicatethatwhilemultipleDSBsincloseproximityarewelltoler-
ated,parallelinductionofDNAbreaksondifferentchromosomesis
highlytoxic.

Multiplexedgenetargetingdoesnotcausewidespreadproxi-
mityeffectsinDrosophila
CRISPR-Casnucleaseactivitycaninduceunintendedon-targeteffects,
including large deletions affecting multiple neighboring genes and
chromosome-arm truncations that remove dozens to hundreds of
genesincis19–21.Sucheventscancause‘proximityeffects’,wheregene
knockoutsdisplayphenotypicsimilaritiestounrelatedgeneslocated
on the same chromosome arm18. We tested whether similar effects
occurduringCas12a-mediatedknock-outmutagenesisinDrosophila.
Because truncations extending from a double-strand break
towardthetelomereaffectmoregeneswhenthebreakoccurscloser
tothecentromere,proximityeffectshavebeenshowntomanifestasa
correlationbetweenphenotypicimpactandchromosomalpositionin
mammaliancells18.Totestifsucharelationshipisalsoobservablein
Drosophila,weperformedCas12a+mutagenesisinwingprecursorcells
using HD12aCFD arrays targeting 387 genes distributed across all
chromosomesandexaminedtheeffectonwingsizeinrelationshipto
targetgeneposition(SupplementaryFig.3).Nocorrelationbetween
phenotypic strength and chromosomal position was observed, sug-
gestingthatchromosome-arm-scaledeletionsdonotoccurfrequently
infliesundertheseconditions.

To increase sensitivity for detecting proximity effects, we per-
formed a focused analysis of gene pairs located in close genomic
proximity. We examined pairs in which one gene produces a well-
characterizedphenotypewhenmutatedandtheneighboringgenehas
notbeenfunctionallycharacterized.Theuncharacterizedgeneswere
located centromeric to the characterized gene, ranging from 0kb
(overlapping on the opposite strand or intronic) to 82kb away. If
deletionsextendingfromthetargetsitefrequentlyaffectneighboring
loci,targetingtheuncharacterizedgeneswouldbeexpectedtophe-
nocopyknockoutsofthecharacterizedgenes.Wefirstexamined10
pairsincludinggeneswithwellestablishedphenotypesinwingmor-
phogenesis.Targetinganyofthe10characterizedgeneswithCas12a+
andHD12aCFDsgRNAarraysproducedhighlypenetrantphenotypes
fullyconsistentwiththeirknownfunction(Fig.4a–j).Theseincluded
strong reductions in wing size in all cases, as well as more specific
phenotypes,suchasafailuretodifferentiatewingveins(Egfr,tkv,smo,
Mad) orlossof margin tissue (wg, Su(H)). In contrast, targeting the
uncharacterizedgenesinclosevicinityresultedineithernoeffector
only mild effects on wing size and did not recapitulate any of the
specificphenotypesobservedwhendisruptingtheneighboringchar-
acterizedgenes(Fig.4a–j).

Wecomplementedthesefindingsbyexamininggenesadjacentto
sepia,ebonyandforked,whichcontrolpigmentationorbristlemor-
phology, respectively. In three of four cases, targeting neighboring
genesproducednoevidenceofproximityeffects(Fig.4k–p).However,
targetingGstO3,whichislocatedinverycloseproximitytotheeye
pigmentationgenesepia, resultedinmild eyepigmentationdefects
withlowpenetrance(13%,n=141,Fig.4k,i).Thesephenotypeswere
lessseverethanthosecausedbycompletesepiadisruption,suggesting
potentialeffectson regulatoryelementsratherthandeletion ofthe
sepiacoding sequence.Notably,theclosesttargetsite in theGstO3
sgRNAarrayislocated just200bp upstream ofthe sepiatranscrip-
tionalstart site and therefore likelyin very closeproximity to such
regulatoryelements.

Intheexperimentsdescribedabove,thecharacterizedgenesare
recessive, meaning that both alleles would need to be affected to
producemeasurablephenotypes.Whilethisappliestomostgenes,a
small minority in the Drosophila genome are haploinsufficient and
wouldthereforebemorevulnerabletoproximityeffects.Totestthis
morestringentscenario,weexaminedtwoHD12aCFDlinestargeting
genesadjacenttoMinuteloci,haploinsufficientgenesencodingribo-
somalproteinsubunits38.DisruptionofasingleMinutealleletriggers
apoptosiswhenmutantcellscontactwild-typecellsinthewingdisc
epithelium39.DirectlytargetingaMinutegeneinwingprecursorcells
resultedinextensiveapoptosisandlethality,withonlyrareescapers
exhibiting severely degenerated wings (Supplementary Fig. 4). In
contrast, targeting either of the two genes proximal to Minute loci
causednooronlyminimalapoptosisintheepithelium,nolethality,
andhadonlyminimaleffectsonwingsizeandmorphology(Supple-
mentaryFig.4).

Taken together, these results demonstrate that multiplexed
Cas12a+genetargetinginDrosophiladoesnotgiverisetowidespread
proximity effects, with effects on neighboring functional elements
occurringonlyatlowfrequencyoronlywhentheseelementsarein
very close proximitytothetargetsite.

Large-scale,unbiasedactivityscreeningrevealshighspecificity
ofmultiplexedCas12a+geneediting
Inductionofoff-targetmutationsrepresentsanotherclassofpotential
unintendedoutcomesofCRISPRgeneeditingexperiments.Giventhe
substantial heterogeneity in off-target propensity among sgRNAs, a
meaningful evaluation of gene editing specificity requires analysis
across large sgRNA collections. However, suitable methods for this
purpose have not been established in Drosophila or most other
organisms.Wethereforesoughttodevelopahigh-throughputassay
for the unbiased detection of CRISPR nuclease activity across large
sections of the genome. We focused on the visualization of loss of
heterozygosity(LOH),acommonrepairoutcomeofCRISPR-induced
chromosome breaks in diverse systems ranging from yeast to
humans21,40–42, and a prevalent consequence of Cas9-mediated DNA
double strand breaks in Drosophila33,43. Two distinct modes of LOH
havebeendescribed:copy-numberneutral(CN-)LOH,whichresults
frominterhomologrecombination,andcopy-numberloss(CL-)LOH,
whichcanarisethroughlargedeletions,chromosometruncationsand
chromothripsis(Fig.5a).Aheterozygousfluorescentreporterenables
visualizationanddiscriminationbetweentheseLOHmodes:CN-LOH
produces equal populations of cells that have gained or lost the
reporter,whereasCL-LOHgeneratesonlyreporter-losscells(Fig.5a,
b).TodeterminetherelativefrequencyofCN-versusCL-LOHinDro-
sophila, we induced double-strand breaks on a reporter-bearing
chromosomearmusingfivedistinctHD12aCFDsgRNAarrays,includ-
ingtwotargetingincloseproximitytothereporter.Quantificationof
fluorescence-gainandfluorescence-losscellsrevealedapproximately
equalratiosforallsgRNAlines,indicatingthatCN-LOHpredominates
inthissystem(Fig.5c,andSupplementaryFig.5).Thedifferencein
prevalenceofLOHmodesbetweenDrosophilaandmammaliancells
likelyreflectsknowndifferencesinchromosomebiology(seeDiscus-
sion) and provides an explanation for the very low frequency of
proximityeffectsobservedintheexperimentsdescribedabove.

LOH detection exhibits strict chromosomal specificity, as
reportersrespondexclusivelytoDNAbreaksinducedbetweentheir
insertionsiteandthecentromere.Areporteronchromosomearm
2L respondsonly toCas12a+ activity directed bysgRNAs targeting
2L,whilea 3R reporter respondsonlytosgRNAstargetingon3R
(Fig. 5d, e). Furthermore, sequencing of target sites from sgRNA
arraysthatinduceLOHatlowfrequencyrevealedthatthisapproach
can detect low Cas12a+ activity, indicating suitable sensitivity for
Drosophila research applications (Supplementary Fig. 6). Collec-
tively, these results establish that LOH reporters enable unbiased
detectionofDNAbreakinductionacrossentirechromosomearms
and provide a sensitive readout for measuring CRISPR nuclease
activity.

To enable high-throughput LOH screening, we constructed
reporter genes expressing fluorophores under a tissue-specific
enhancerthatrestrictsexpressiontolarvalwingandhaltereimaginal
discs44(Fig.5fandmethods).ThisenablesvisualizationofLOHinliving
animalswithaconventionalfluorescentstereoscope(Fig.5g,h).We
appliedthissystemtoscreenforLOHacrossbotharmsofchromo-
some 2, using hundreds of HD12aCFD lines encoding over 2000
unique sgRNAs. These screens monitored CRISPR activity over
44.6Mb,representing33%oftheDrosophilagenome.

First,wefocusedonsgRNAarraysdesignedtotargetgeneson
other chromosome arms or distal to the reporter insertion site.
BecauseindividualsgRNAsaresufficienttoinducerobustLOH(Sup-
plementaryFig.7),anyarray-encodedsgRNAswithoff-targetactivity
on the screened chromosome arm are expected to lead to repro-
ducibleLOH.Thescreenon2Lincluded1769suchsgRNAs,whilethe
2Rscreencomprised1605.WedetectednoLOHwithsuchsgRNAson
2RandsporadicLOHin16casesinthescreenon2L(Fig.5i,j).The
lattereventscouldnotbereproduceduponrescreeningwithlarger
samplesizes(SupplementaryFig.8),suggestingtheylikelyrepresent
eitherstochasticbackgroundLOHorscreeningartefacts,althoughwe
cannotexcludethepossibilityofoff-targetactivityoccurringatvery
lowfrequency.

We then analysed the results from LOH screening with the
HD12aCFDarraystargetinggeneslocatedbetweenthereporterinser-
tionsitesandthecentromere.Thesewere81lineson2Land88lines
on2R.OnlyasinglelinefailedtoinduceLOH(Fig.5i,j),demonstrating
highlyrobuston-targetactivityofHD12aCFDarrays.

Insummary,LOHvisualizationenabledustoscreenforCas12a+
activitywithover2000sgRNAsacross33%oftheDrosophilagenome.
Thisconstitutes,toourknowledge,thesofarlargestunbiasedscreen
fornucleaseactivityconductedinvivo.Itrevealedon-targetactivity
forover99%ofHD12aCFDarraysanduncoveredpotentialoff-target
activityinlessthan1%ofsgRNAs,indicatingexcellentefficiencyand
specificityofgenetargetingwithHD12aCFDarrays.

MultiplexedCas12a+genetargetingoutperformsestablished
Cas9-basedmethods
We next compared the efficiency of gene disruption with Cas12a+
andHD12aCFDarraystothatofcurrentstate-of-the-artCas9-based
methods.First,weperformedubiquitousortissue-specificCRISPR
mutagenesis of endogenous genes with known loss-of-function
phenotypes.Weexaminedwhite(w)disruptioninneuronalphoto-
receptors,wherelossoffunctionleadstoeyepigmentationdefects.
Strikingly, HD12aCFD-mediated gene targeting achieved near-
complete w disruption (96±5% white-eyed tissue), whereas Cas9
withapubliclyavailablesgRNAgeneratedhighlymosaiceyeswith
only 50±26% loss of pigmentation (Fig. 6a, b). We extended this
comparisontotheintestinalepitheliumbyinducingmutagenesisof
Notchandneuralized,whicharebothcriticalregulatorsofintestinal
stemcellproliferation.HD12aCFDtargetingofeithergeneresulted
in significantly greater expansion of mitotic cells compared to a
dual-sgRNACas9system(Fig.6c,d).Similarly,targetingsmoothened
(smo) specifically in the pouch domain of wing imaginal disc epi-
thelia resulted in near complete loss of Smo protein with a
HD12aCFDline,whilecellsexpressingtwosgRNAsandCas9retained
asignificantnumberofSmopositivecells(Fig.6e,f).Collectively,
thesedataindicatethatgenetargetingwithCas12a+andmultiplexed
sgRNAsachievessuperiorgenedisruptionefficiencyacrossdiverse
tissues.

To systematically benchmark HD12aCFD performance, we
developed a quantitative assay using adult wing size as a scalable
phenotypic readout. We targeted genes specifically in wing pre-
cursorcellsusingpdm2-Gal4and employedautomatedimageana-
lysiswithacustom-trainedsegmentationalgorithmtomeasurewing
sizeacrossmultipleindividualspergenotype(SupplementaryFig.9).
Applyingthissystemtoover300targetgenes,HD12aCFD-mediated
targeting of known growth regulators consistently produced size
reductions, while targeting of control genes left wing morphology
intact, confirming both efficiency and specificity (Supplemen-
taryFig.9).

We next compared HD12aCFD arrays against existing CRISPR
resourcesforgenedisruptioninDrosophilathroughparallelscreening.
We evaluated two major collections: sgRNA lines from the Bloo-
mingtonDrosophilaStockCenter(BDSC)andtheHD_CFDlibraryfrom
theViennaDrosophilaResourceCenter(VDRC)10–12.Thesecollections
encompass diverse design strategies, including different promoter
choices(U6:3orUAS)andsgRNAnumbers(oneortwo),representing
therangeofeditingapproachescurrentlyinuse.Fordirectcompar-
ison,weobtainedallavailableBDSCandVDRClinestargetinggenes
also targeted by HD12aCFD lines and performed side-by-side wing-
specificmutagenesis.

HD12aCFD arrays significantly outperformed both existing
resources when tested against 154 BDSC and 103 VDRC lines
(p<0.001,Wilcoxonsigned-ranktest;Fig.6g,h).Asexpected,many
genesshowednowingphenotypewitheithersystem,indicatingthat
thesegenesarenotrequiredforwingdevelopment.Amonggenesthat
producedphenotypesandthusprovideinformativecomparisonsfor
assessing knockout efficiency, effects ranged from comparable
between systems to substantially stronger with HD12aCFD arrays.
Importantly,thisincludedseveralestablishedwinggrowthregulators
suchaswg,ap,tkv,Su(H),Raf,andCdk1,whichproducedminimalto
moderatephenotypeswithCas9-basedsystemsbutresultedinstrong
sizereductionswithHD12aCFDarrays.Thisdemonstratesthatthese
outliers represent functional false negatives in conventional Cas9-
basedscreeningapproachesandestablishesHD12aCFDtechnologyas
asubstantialadvanceforrobustgenedisruption.

Finally, we tested whether a recently developed Cas9 sgRNA
expressionvectorfeaturinganoptimizedscaffoldandefficienttRNA-
based processing45 could close the performance gap between Cas9
andHD12aCFDsystems.Sincethisimproveddesignhasnotyetbeen
incorporatedintothelarge-scaleresourcesevaluatedabove,wetested
it separately on three morphogenetic regulators where HD12aCFD
arrays had faithfully recapitulated expected loss-of-function pheno-
types, while HD_CFD lines produced weaker effects. We cloned the
samesgRNAsusedintheHD_CFDlinesintotheimprovedexpression
vectorandinsertedthematthesamegenomiclandingsite,eliminating
sgRNAselectionandexpressionasaconfoundingvariable.Whilethe
improvedvectormodestlyenhancedphenotypicpenetrancefortwo
ofthreegenescomparedtoHD_CFDlines(SupplementaryFig.10),it
stillfellsubstantiallyshortofHD12aCFDperformanceacrossalltar-
gets. These controlled comparisons demonstrate that HD12aCFD
technology surpasses even the latest optimizations in Cas9-based
systems.

Increasedgenedisruptionefficiencyrevealsasomaticfunction
fortradeembargo
Themutagenesisscreensdescribedabovealsoincludedgeneswith
nopreviouslydocumentedroleinwingmorphogenesis.Oneofthem
wastradeembargo(trem),whichencodesaC2H2zinc-fingerprotein.
Mutagenesis of trem with a HD12aCFD sgRNA array resulted
in a 24.5±3.8% reduction in wing size, while gene targeting
withaHD_CFDsgRNApairproducednosuchphenotype(4.3+−4.9%
reduction in wing size, Fig. 7a, b). Additionally, we observed a
highly penetrant defect in wing vein differentiation in animals
expressingCas12a+andHD12aCFD-trem,characterizedbyabsenceof
the posterior cross vein and defects in the L2, L4, and L5 veins
(Fig.7a”,c,d).Nosuchveindefectsweredetectedwhenmutagenesis
was performed with Cas9 and the HD_CFD-trem sgRNA construct
(Fig.7a’,c,d).

The wing phenotypes observed exclusively after multiplexed
Cas12a+ targeting of trem could result from either more efficient
gene disruption or potential artifacts associated with the
HD12aCFD-tremline.Todistinguishbetweenthesepossibilities,we
generatedtwoadditionalCas9sgRNApairs.Thesenewconstructs
produced wing size and vein defects when combined with tissue-
specific Cas9 expression (Fig. 7b–f). Although these phenotypes
were less severe than those observed with HD12aCFD-trem, they
recapitulatedallaspectsofthewingphenotype:sizereduction,loss
of the posterior cross vein, and defects in the lateral longitudinal
veins. These results demonstrate that the observed phenotypes
arise from trem disruption and are not specific to any particular
sgRNAline.

Arolefortreminwingmorphogenesiswassurprising,asitwas
notreportedinapreviousstudyfocusingonthefunctionoftremin
Drosophila46. This study analysed a transposon insertion allele,
tremf05891, which was classified as loss-of-function based on unde-
tectableTremproteinlevelsusingaspecificantibody.However,the
tremcodingsequenceremainsintactintremf05891,raisingthepossi-
bilitythatinsteaditcouldbeastronghypomorphicallele(Fig.7g).
WethereforegeneratednewtremallelesthroughCRISPRgenetar-
geting in the germline. We recovered two indel alleles in the trem
coding sequence that disrupt the protein reading frame. After
backcrossing both alleles for multiple generations to eliminate
potentialsecondarymutations,wecombinedeachallelewithabal-
ancer chromosome carrying a dominant marker and tested their
homozygousviability.Incontrasttotremf05891,whichishomozygous
viable without somatic phenotypes, both trem CRISPR alleles are
homozygous lethal, with animals lacking balancer chromosomes
dyingatmid-larvalstage(Fig.7h).

Together, these findings establish trem as an essential gene in
Drosophila with critical roles in growth and patterning of wing
appendages. These functions remained undetected with existing
germlineallelesandanestablishedCas9sgRNAline,asthesetoolsdid
not achieve sufficient levels of gene inactivation to reveal the full
spectrumoftremactivities.

Methods
Plasmids
Plasmid construction. Plasmid cloning was performed using PCR
ampliconsgeneratedwithQ5PCRpolymerase(NewEnglandBiolabs).
DNAfragmentswereassembledbyeitherIn-Fusioncloning(Takara)or
byGoldenGatecloningusingBbsI-HFandT4DNALigase(bothNew
England Biolab) using standardprocedures or byextension overlap
PCR (see below). PCR conditions followed manufacturer’s recom-
mendations. Individual oligonucleotides for cloning were ordered
from Integrated DNA Technologies in standard, desalted format.
NewlyclonedinsertswereconfirmedbySangersequencing.Sequen-
cesofplasmidsandprimerscanbefoundinSupplementaryData2.
PlasmidsareavailablefromtheEuropeanPlasmidRepository(https://
www.plasmids.eu) and Addgene (https://www.addgene.org) with
accessionIDsprovidedbelowforeachplasmid.

pSTAR(1–6x).Thereporterplasmidsformultiplexedcutting(sgRNA
targetsitearray(STAR))weregeneratedbycloningafragmentofthe
lacZcodingsequenceintoaplasmidbackbonecontainingAmpRand
mini-whiteselectiongenes,aswellasattBandori,thatwasderivedby
BamHIdigestionofplasmidpCFD64.ThelacZfragmentlacksbotha
promoter and start codon and is presumed to be transcriptionally
inactive. Afterwards, the sequence TTTCCAGGGAACTCCCATCCACC
ATGGCTGGTATTCC, which can be targeted by a Cas12a sgRNA
(CAGGGAACTCCCATCCACCATGG)orCas9sgRNA(GAACTCCCATC-
CACCATGGC), was introduced by overlap extension PCR. Two PCR
ampliconsweregeneratedwithanoverlappingsequencecontaining
thesgRNAtargetsite(seeSupplementaryData2forprimersequen-
ces).EquimolaramountsofthetwoPCRampliconsweremixedand
subjectedtoPCRfor15cycles.Then,primersannealingtotheendof
thefusedPCRampliconwereaddedandfulllengthproductsamplified
for another 20 PCR cycles. PCR amplicons were separated on a 1%
agarosegel,fulllengthampliconswereexcised,purifiedandcloned
intoBamHIdigestedpCFD6byIn-Fusioncloning.

act5C-mScarlet. A double-stranded DNA fragment encoding the
act5Cpromoter,mScarletcodingsequenceandact5C3’UTRwassyn-
thesizedfromIntegratedDNATechnologies.Itwasassembledwitha
plasmidbackbonefragmentcontainingAmpRandmini-whiteselection
genes,aswellasattBandori,whichwasobtainedbyBamHIdigestion
ofplasmidpCFD64byIn-Fusioncloning.Theplasmidisavailablefrom
the European Plasmid Repository (EPR#897) and Addgene (Plasmid
230906).

GMR11F02-Gal4UAS-GFP.AfragmentencodingtheGMR11F02reg-
ulatory element and Gal4 coding sequence was PCR amplified with
primers GMR11F02G4fwd and GMR11F02G4rev from plasmid
pGMR11F01-Gal4(agiftfromRyanLokerandRichardMann,Columbia
University,New York).The fragment was then inserted into pUAST-
EGFPdigestedwithXhoIbyIn-Fusioncloning.Theplasmidisavailable
from the European Plasmid Repository (EPR#898) and Addgene
(Plasmid230907).

UAST-uMCas12a+nls1x.PlasmidUAST-uMCas12a+nls1xwasderivedfrom
UAS-LbCas12a25.AnupstreamopenreadingframewasPCRamplified
fromUAS-uMCas910withprimersuMCas12afwdanduMCas12arevand
gelpurifiedandclonedintoUAS-LbCas12adigestedwithEcoRIbyIn-
Fusioncloning.

UAST-FRT-GFP-FRT-uMCas12a+nls1x. Plasmid UAST-FRT-GFP-FRT-
uMCas12a+nls1xwasderivedfromUAST-uMCas12a+nls1x.Theplasmidwas
digested with EcoRI-HF and HindIII and the sequence encoding the
FRT-GFP-FRTcassettewassynthesizedbyIntegratedDNATechnolo-
giesandinsertedbyIn-Fusioncloning.Theplasmidisavailablefrom
the EuropeanPlasmidRepository(EPR#899) and Addgene (Plasmid
230908).

UAST-uMCas12a+nls2x.PlasmidUAST-uMCas12a+nls2xwasderivedfrom
UAST-uMCas12a+nls1x.ThesequenceencodinganadditionalC-terminal
nucleoplasmin nuclear localization signal was ordered as double-
stranded DNAfrom Integrated DNATechnologies and inserted into
UAST-uMCas12a+nls1xdigestedwithNdeIandHpaIbyIn-Fusioncloning.
The plasmid is available from the European Plasmid Repository
(EPR#900)andAddgene(Plasmid230909).

pGRACE.PlasmidpGRACEwasderivedfrompAct5c-LbCas12a+29.The
backbonewaslinearizedwithEcoRIandXhoI.Thesequenceencoding
the uORF with binding sites for Cas12a and Cas9 sgRNA flanked by
microhomologyandthefluorescentproteinwassynthesizedbyInte-
grated DNA Technologies and inserted by In-Fusion cloning. The
plasmidisavailablefromtheEuropeanPlasmidRepository(EPR#901)
andAddgene(Plasmid230910).

CloningofsgRNAplasmids.Individual sgRNA plasmids were con-
structedaspreviouslydescribed29.Briefly,Cas12asgRNAarrayswere
clonedintotheBbsIsiteoftheexpressionvectorpCFD8(European
Plasmid Repository EPR#32; Addgene #140619). For individual
sgRNAs the spacer sequence was ordered as two individual oligo-
nucleotides encoding the top and bottom DNA strand, including
appropriate overhangs, phosphorylated, annealed and ligated into
pCFD8. To clone individual plasmids encoding sgRNA arrays, the
array sequence was split into several oligos, with the split being
locatedinthemiddleoftheuniquespacersequences.Oligoswere
phosphorylated, assembled with ‘bridge’ oligos, which are reverse
complementarytothejunctionsoftwooligos,andligatedbyT4DNA
ligase(NewEnglandBiolabs).Subsequently,ligatedoligosencoding
thefullsgRNAarraywereamplifiedbyPCRandclonedintotheBbsI
siteofpCFD8byGoldenGatecloning.Wehavepublishedadetailed
step-by-step cloning protocol in the supplementary material of
reference29. The spacer sequences of sgRNAs are listed in Supple-
mentaryData1.

sgRNAlibrarydesign
HD12aCFD sgRNAs have been designed using the CRISPR Library
Designer(v.1.5)32.GuidesweredesignedagainsttheD.melanogaster
genomereleaseBDGP6.22,allowingonlyguidesincodingsequences
withlessthan10predictedoff-targetsofaneditdistancelargerthan
2. For aligning crRNA target sites back to the reference genome
Bowtie was used in very-sensitive mode and the first 3 PAM distal
baseswereignored.ThePAMrequirementwassettoa5’adjacent
TTTN.AsbasisfordesignservedalistofENSEMBLidentifierscov-
ering all protein coding genes in BDGP6.22. Raw, designed crRNA
targetswerethenfurtherprocessedinacustomRpipelinetoyield
optimized crRNA target pairs for combinatorial mutagenesis as
follows.

TheCLDoutputfileswerefilteredaccordingtothedesigncriteria.
Inthefollowingsteps,relevantcolumnsfromthelargeCLDoutput
tablewereselected.Everythingirrelevanttothelistoftargetgeneswas
removed,andthe‘NumberofHits’columnwasadjustedtorepresent
off-target count by subtracting one count. After that, all sequences
containingBbsI(GAAGAC)andBsaI(GGTCTC)restrictionsites,non-
uniquecrRNAtargets,andcrRNAtargetsadjacenttoaTTTTPAMwere
removed.AdditionalselectioncriteriafortheremainingpoolofsgRNA
designswereannotatedasfollows:thenumberoftargetedprotein-
coding transcripts, whether the sgRNAs target the first half of the
target gene, and the GC content of sgRNA sequences. Only those
sgRNAsthathitatleastmax(#transcripts)-1transcriptswerekept,and
theaveragegeneexpressionforeachtranscripthitbyaputativecrRNA
wasannotated.Designsthatunintentionallyalsohitacodingsequence
ofadifferentgenethantheyweredesignedforwereremoved.Further,
smallgenesthatarecontainedinoverarchinggiantgenes,likekirre,or
Raf,wereexcluded.EachsgRNAwasannotatedonhowmanyCDSit
targetsandifittargetsinthefirsthalfofthetargetgene.Tocheckthe
poolforsanity,thesgRNAswerewritteninGFF3format.Thisfilecould
thenbeloadedintoanarbitrarygenomebrowser.Next,180random
pairsofsgRNAsweredrawnforeachgeneandscoredbyoptimizing
pairdistance,microhomologyscore59,exonexpression,isoformcov-
erage,off-targetcounts,GCcontent,andexonpositionrelativetothe
transcript.Thetwotop-scoredcrRNApairswerechosenforfurther
qualitycontrolsbymanualandautomaticinspection.

CloningoftheHD12aCFDsgRNAlibrary
Cas12asgRNAarrayswereclonedintotheBbsIsiteoftheexpression
vector pCFD8 (European Plasmid Repository EPR#32; Addgene
#140619), which has been previously described29. Oligonucleotides
encodingfoursgRNAs,aswellasprimerbindingandBbsIrestriction
sitesateitherendweresynthesizedbyTwistBioscience.Theoligo-
nucleotidedesigncanbefoundinSupplementaryData2.Oligoswere
resuspended in dH O and amplified by PCR using primers Cas12ar-
2
rayAMPfwd2andCas12arrayAMPrev2.PCRproductswereseparatedon
a 1% agarose gel, excised, purified and cloned into pCFD8 by Gold-
enGatecloningusingstandardconditions.DNAwastransformedinto
chemicallycompetentE.coli(StellarSilvercompetentcells,Takara)
and plated on LB agar plates containing Carbicilin. After overnight
incubationat37C,tenindividualcolonieswerepickedforvalidation
purposesandallothercolonieswerepooled.DNAwasextractedusing
QIAprep Spin Miniprep Kit (Qiagen) following manufacturer’s
instructions. DNA from individual colonies was validated by Sanger
sequencing. When at least 9 of the 10 tested colonies contained
unique,correctsgRNAplasmidsweproceededwithinjectionofthe
DNA from the pooled colonies into nos-PhiC31; attP40 transgenic
Drosophilaembryos.

VisualizationofCRISPR-editedPCRamplicons
To visualize diversification of the target locus by CRISPR editing,
reflecting the heterogeneityofthe edited sequences, genomic DNA
wasextractedfromindividualfliesaspreviouslydescribed29.Flieswere
collected in PCR tubes containing 30µL squishing buffer (10mM
Tris⋅HClpH8,1mMEDTA,25mMNaCl,200µg/mLProteinaseK).Flies
weredisruptedinaBeadRuptor(Biovendis)for20sat20Hz.Samples
werethenincubatedfor30minat37°C,followedbyheatinactivation
for3minat95°C.Typically,2µLofsupernatantwereusedin30-µL
PCR reactions. The target locus was amplified by PCR using locus-
specific primers (Supplementary Data 2) with Q5 High-Fidelity DNA
Polymerase(NewEnglandBiolabs)andnomorethan30PCRcycles
understandardconditions.Ampliconswereseparatedon1%agarose
gels.NotethatampliconpoolscontainingdiverseCRISPReditstypi-
callyformheteroduplexesduringPCR,whichresultsintheformation
ofasmeartowardshigher-andlowermolecularweightproductson
thegel.

IdentificationofCRISPRallelesbySangersequencing
To identify individual CRISPR-induced alleles mutagenesis was per-
formedinthegermlineandgenomicDNAwasextractedfromindivi-
dualoffspringasdescribedaboveandthetargetlocuswasamplified
by PCR using locus-specific primers (Supplementary Data 2) that
annealedtypically300–500bp5’and3’ofthesgRNAtargetsite.PCR
amplicons were purified using paramagnetic beads60 and sent for
Sanger sequencing (Eurofins Genomics). Reads were only excluded
fromanalysisifsequencingqualitywasinsufficientforunambiguous
basecalling.

Flystocksandhusbandry
The list of target genes and corresponding sgRNAs for which we
generated HD12aCFD transgenic lines is reported in Supplementary
Data1.AllothertransgenicDrosophilastrainsusedorgeneratedinthis
studyarelistedinSupplementaryData3.Flystocksaremadepublicly
availablethroughthenon-profitViennaDrosophilaResourceCenter
(https://vdrc.at).Drosophilawereculturedonstandardcornmealfood
mediainanincubatorsetto27°Cwitha12hlight−12hdarkcycleand
60%relativehumidity.Adultfliesweretransferredtofreshfoodvials
every3–4days.

Transgenesis
Transgenesis was performed with the PhiC31/attP/attB system and
plasmids were inserted in landing sites on the second or third
chromosomeasindicatedforeachline.Microinjectionofplasmids
intoDrosophilaembryoswascarriedoutusingstandardprocedures.
TransgenesisofcrRNAplasmidswastypicallyperformedbyapooled
injection protocol, as previously described29. Briefly, individual
plasmidswerepooledatequimolarratioandDNAconcentrationwas
adjusted to 250ng/μL in dH O. Plasmid pools were microinjected
2
into blastoderm embryos, raised to adulthood and individual flies
crossed to P{ry[+t7.2] = hsFLP}1, y[1] w[1118]; Sp/CyO-GFP or
P{ry[+t7.2] = hsFLP}1, y[1] w[1118];; Sb/TM6B. Transgenic offspring
were identified by eye color and individual transgenic flies from
pooledplasmidinjectionsweregenotypedaspreviouslydescribed24.
Briefly,genomicDNAwasextractedfromindividualfliesasdescribed
above and the sgRNA locus was amplified by PCR using primers
(pCFD8genofwd5: TTAACGTCGGGGCTTTGAGT,pCFD8genorev6:
CGACACTAGTGGATCCGTTGT).Flieswerecrossedtobalancerflies,
andstabletransgenicstocksweregenerated.

Immunofluorescencestaining
Drosophila larvae were dissected in ice-cold PBS and fixed in 4%
paraformaldehydefor25minutesatroomtemperature.Tissuewas
washedwithPBT(PBSwith0.3%Triton-X100)andblockedwith1%
heat-inactivatednormalgoatseruminPBT.Antibodieswerediluted
inPBTandtissuewasincubatedovernightat4°C.Antibodiesused
wereanti-cleavedDCP1(CellSignaling,CAT9578S,diluted1:600),
anti-phosphorylated Histone 3 (Cell Signaling, CAT 9701, 1:1000),
anti-LbCas12a (Diagenode, CAT C15310263, 1:800), and anti-
Smoothened(DSHB,CAT20C6,1:200).AfterserialwashesinPBT,
tissue was incubated in a secondary antibody (labeled
with Alexa Fluorophores, diluted 1:600, Life Technologies) and
Hoechst(diluted1:2000)inPBTfor2hatroomtemperature.Fol-
lowing serial washes tissue was mounted in Vectashield (Biozol
Diagnostica).

Imaging
TissuewasimagedonaLeicaSP8laser-scanningconfocalmicroscope
withanoil40x/NA1.4lensusingthesequentialscanningmodeora
Keyence BZ-X810 fluorescent microscope with an 20x airobjective.
ImageprocessingandanalysiswereperformedwithFIJI.Imagingof
adult flies or wings was performed with a Leica M165 FC stereo-
microscopeequippedwithaLeicaDFC295camera.

Lossofheterozygosityscreening
LOH wasmonitored using heterozygous transgenes encoding fluor-
escent proteins. Upon LOH of the chromosome arm on which the
transgene is located, cellsgain or loose fluorescence, whichcan be
visualizedusingafluorescencemicroscopeorstereoscope.Plasmids
wereinsertedinattPsitesatthedistalendofthechromosome,either
atcytogeneticposition22Conchromosomearm2L,59Donarm2R,
orat99Fonarm3R.Animalswerealsotransgenicforact5C-Cas12a+
andHD12aCFDsgRNAs,andLOHwasassessedinwingimaginaldiscsat
larval 3rd instar wandering stage. We confirmed that LOH is also
readilydetectedinothertissues,suchasthebrainandtheintestine.
ToassayLOHwithhighresolutiontheact5C-mScarletplasmidwas
used,whichresultsinubiquitousandrelativehomogenousexpression
ofmScarlet.Thirdinstarlarvaeweredissectedonice,andwingdiscs
werefixedin4%paraformaldehydeinPBSatroomtemperaturefor
25minutes.Nucleiwerestainedwith1µg/mlHoechstfor20minutes,
anddiscsweremountedinVectashieldmountingmedium.Imaginal
discs were imaged with a Leica SP8 laser-scanning confocal micro-
scope with an oil 40x/NA1.4 lens. Images were analyzed in Fiji by
outlining all areas that lost or gained fluorescence and using the
measurefunctiontoquantifytheirsize.Notethattheareathatcells
with LOH occupy in the tissue reflects both the frequency of LOH
(numberofcellsthatgainorlosereporterexpression)andthetime
pointofoccurrence(numberofcelldivisionsafter LOH).Whilethe
act5C-mScarletreporterisidealtoobserveLOHinisolatedtissuesby
high-resolution imaging, the ubiquitous reporter cannot reliably
detectLOHinwholeanimals,astheirmultilayeredtopologyobscures
LOHeventsinindividualtissues.

For high-throughput LOH screening in living animals, a
GMR11F02-Gal4UAS-GFP(forthescreenon2L)oraGMR11F02-Gal4
UAS-mScarletreporter(for2R)wasused.GMR11F02-Gal4drivesspe-
cificexpressioninthepouchregionofthewingandhaltereimaginal
discs.Larvaewereimmersedinadropoftapwaterandobservedunder
aLeicaM165FCfluorescentstereomicroscopewith6.3xmagnifica-
tion.Toincreaseconsistency,eachscreenwasperformedbythesame
observer(F.P.screened2L,whileE.P.screened2R),whowasblinded
to larval genotypes. The fluorescent signal from the GMR11F02
reporterinthediscpouchshowsnaturalvariation,withstrongersignal
typicallyobservedatthedorsaledgeoftheexpressiondomainand
weakerfluorescencealongthedorsal-ventralboundary.Therefore,the
observerfocusedonlargerpatchesofcellsthatlostfluorescenceand
scoredthelevelofLOHperwingdiscusingthefollowingscale:0-no
LOHobserved;1-oneortwosmallclones;2-onetothreesmallto
largeclones;3severallargeclones.ThemeanLOHscorepresentedin
thefiguresrepresentsthemeanscoreofallwingdiscs(typically10−
20)recordedforaparticulargenotype.

Drosophilawingsizephenotyping
DissectedDrosophilawingsweremountedonglassslidesandimaged
usingaLeicaM165FCstereomicroscope.Imageswereusedtoretrain
Cellpose’s cyto2 model61 using human-in-the-loop capabilities and
parameters:diameter140,flow1,andcellprob.1.Usingthismodel
(‘fly_wings’)wingsweresegmented,outlineswereexportedtoFijiand
wingsizewasmeasuredusingtheMeasurefeature.ThecustomCell-
posemodel(“Wingpose”)isprovidedinSupplementaryData4.

Generationoftremalleles
Germlinemutationsintremweregeneratedaspreviouslydescribed62.
Briefly,nos-Gal4VP16UAS-uMCas12a+flieswerecrossedtoywhs-Flp;
HD12aCFD0127sgRNAflies.Maleoffspring,expressingCas12a+inthe
germlineandtremspecificsgRNAs,werethencrossedtow*,+,+virgin
femalesandoffspringharboringmutationsintremwereidentifiedby
genotypingPCRusingprimerstremgenofwd1andtremgenorev1.Two
mutant alleles were recovered: tremdel10 harboring a ten-base pair
deletion(CCTGAACAATCCCAG - del10 - GAGCTGCTCCACGAC) and
tremdel11 (TTGCCTGAACAATCC- del11 - AGGAGCTGCTCCACG).Both
shiftthereadingframeinthefirstcodingexon,resultinginatrunca-
tion of about 90% of the coding sequence. Mutant flies were then
backcrossed against w*,+,+ animals for five generations to remove
potentialsecondarymutations.Afterwards,astablestockwasgener-
ated using a y w hs-Flp;; MKRS/TM6B balancer stock. y w hs-Flp;;
tremdel10/TM6B and y w hs-Flp;; tremdel11/TM6B stocks both retainthe
balancerchromosome,ashomozygoustremmutantanimalsdieinmid
larvalstage.

Dataanalysisandvisualization
DataanalysiswasperformedinRusingtheTidyversepackage.Graphs
werecreatedusingggplot2.Microscopeimageswerepreparedusing
Fiji.FigureswerecreatedusingAffinityDesignerV1.

Statisticsandreproducibility
Sample sizes were not predetermined by statistical methods, but are
basedonestablishedpracticesinthefieldtoensuresufficientpowerto
detectphenotypiceffects.Experimenterswereblindedtotheidentityof
theanalyzedgenotypesduringtheLOHscreensreportedinFig.5iandj.
Noblindingwasusedduringallotherexperiments.Statisticalanalysis
wasperformedinRandthedetailsofthestatisticaltestsaregivenin
the figure legends. All experiments, except the large-scale screens
reportedinFigs.5i,5j,6g,6h,wererepeatedmultipletimeswithcon-
sistentoutcomes,supportingtherobustnessandreproducibilityofthe
findings.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Following on from the initial review of the manuscript, the primary concern raised by this reviewer was the possibility of chromosome-arm scale deletions induced by clustered Cas12a DSBs leading to copy number-loss Loss of Heterozygosity (CL-LOH), which would produce confounding proximity based phenotypic effects and limit the efficacy of this approach as a novel gene knockout strategy. Upon reading the revised manuscript, this reviewer commends the authors for embracing this concern and going above and beyond to address it. The experiments in the revised manuscript thoroughly demonstrating an absence of proximity based phenotypic effects from gene knockouts, and the presence of copy number gain events strongly support a predominance of DSB induced CN-LOH and a relative absence of DSB induced CL-LOH events. Moreover, the authors also produced a supporting figure showing that RNAi mediated trem knockdown also shows a similar, albeit weaker wing phenotype as the HDCas12aCFD mediated trem knockout, which ameliorate concerns regarding the novel proposed function of trem. This reviewer therefore recommends publication, notwithstanding a few minor comments that can be addressed with a few small changes in the manuscript text, which are as follows:\n1) The copy-number neutral LOH (CN-LOH) events in the paper are ubiquitously described as mitotic recombination events. While it is true that allele specific Cas9 DSBs induce substantial levels of mitotic recombination associated with crossing over in Drosophila (PMID: 33750946), an important difference is that DSBs induced in this paper produce breaks on both homologues. This raises the formal possibility that CN-LOH events instead occur via reciprocal end-joining events, which have been demonstrated in human HT1080 cells, a p53-positive fibrosarcoma cell line (PMID: 32873648). The reviewer does acknowledge that frequent mitotic recombination has been demonstrated before in Drosophila, nevertheless acknowledging the possibility of reciprocal end-joining events covers all bases.\n2) The method of tracking LOH on chromosome arms with integrated fluorescent reporters to detect off-target genome editing is indeed very convenient, but this approach is limited to tracking events only on the marked chromosome arms. This caveat is usually not acknowledged in the text, rather the phrase “off-target activity” is used without any qualifiers. It is therefore recommended that the text in lines 91 and 420 be changed to “off-target activity on the two chromosome arms with LOH reporters”.\n3) Figure 3 legend line 244 refers to supplementary figure 1, when it seems it should refer to supplementary figure 2.", "reviewer_2": "The authors have done a very nice job revising the manuscript by adding substantial new results. These new results and revisions of text addressed my previous concerns. The revised manuscript is a very nice piece of work which represents a significant advancement in CRISPR technology and application in Drosophila. I am supportive of its publication by Nature Communications but with two minor comments.\n1. Figure 2 characterized Cas12a+ activity using the GRACE reporter. UAS-Cas12a+ appears to have low levels of leaky activity in the wing disc. How is it compared to the leaky activities of previous UAS-Cas9 strains generated by the same group? This information would be useful for people deciding between the Cas9 and Cas12a+ system.\n2. It is not clear how the sequencing results in Supplementary Figure 6 were generated. Were they derived from PCR from a single animal or a mixture of multiple larvae? Or were they from dissected larval wing discs?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have addressed most of my comments. I don't have any further questions."}, "author_response": "We thank all four reviewers for their constructive and insightful feedback on our manuscript.\nThe points you raised have been addressed through extensive additional experimental work,\nas detailed in our responses below. We believe these revisions have substantially\nstrengthened the study and hope you will agree that the manuscript is now suitable for\npublication.\nAll original reviewer comments are copied below with our replies in bold.\nReviewer #1 (Remarks to the Author):\nKnocking out genes via CRISPR-Cas9 catalyzed DNA double-strand breaks (DSBs) is a widely used\napproach for functional studies of gene function in various biological model systems. The principal\nadvantage of this method is the theoretically unlimited flexibility of targeting any part of the genome\nthat is adjacent to a PAM site, with the optimal outcome mostly being small indels generated by\nDNA-DSB repair that disrupt the coding sequence. However, this process is limited in practice by\nsub-optimal sgRNAs, sub-optimal target sequence context and chromatin and transcriptional state of\ntarget sequence, to name a few (PMID: 38440123). An additional drawback of CRISPR-Cas9\nmediated mutagenesis is the possibility of off-target effects, which are quite difficult to detect (PMID:\n36970624), but can create mutations at unintended target sites that may affect functional studies.\nStill another potential pitfall of knocking-out genes with CRISPR-Cas9 catalyzed DNA DSBs is the\npotential of un-intended outcomes at the target site, such as large deletions (PMID: 30010673,\nPMID: 35701408 and PMID: 39496933) and even mega-base scale losses and rearrangements that\ncan truncate large portions of chromosomes, lead to translocations, whole chromosome losses\n(PMID: 35773341, PMID: 34389729, PMID: 33125898, PMID: 34050011, PMID: 37794590, PMID:\n37429857 and PMID: 39386534) and even chromothripsis (PMID: 33846636). Such large genomic\nrearrangements may result in loss of heterozygosity (LOH) over large regions of the targeted\nchromosomes (PMID: 37429857 and PMID: 39386534), which also have the potential to create\nconfounding effects for functional studies involving gene knockout. This was reported in a recent\nstudy by Lazar et al. (PMID: 38811841), where they showed proximity biases where CRISPR\nknockouts show unexpected phenotypic similarities to unrelated genes on the same chromosome\narm. This “proximity effect” is in fact widespread in CRISPR-Cas9 genetic screens and has led to\nmisappropriation of function to genes in large datasets like DepMap, and Lazar et al. proposed the\napplication of a geometric correction algorithm to potentially remove erroneous assignment of\nfunction to genes from large CRISPR-Cas9 screens. In the opinion of this reviewer, “on-target” toxic\neffects such LOH are as much of a concern with CRISPR-Cas9 or any other DNA DSB mediated\nmutagenesis application as off-target effects, if not more, because they can affect Megabases of\nDNA sequences than span over entire chromosome arms and hundreds to thousands of genes.\nIn this study, Port et al. describe a genome editing strategy that combines an enhanced variant of\nCas12a combined with an array of 4 sgRNAs to generate a more efficient gene targeting system\nthan Cas9 based methods. Their experiments demonstrate that their system, referred to in the paper\nas HD12aCFD, routinely leads to stronger DNA target ablation and more penetrant phenotypes in\nvivo in live fruit flies compared to a Cas9 based system with 2 sgRNAs per target (referred to as\nHD_CFD). In addition, the authors assayed for LOH with an extensive number of sgRNA arrays in\nfruit flies with telomere proximal fluorescent reporters on 2L and 3R, and showed that many of these\ncause reproducible levels of LOH specifically on the targeted chromosome arm. These LOH events\nspan several Megabase pairs, in agreement with several studies not only in mammalian cells (cited\nabove), but also in Candida parapsilosis (PMID: 36416551) and tomato (PMID: 37497643). Finally,\nthe authors showed more experiments that reiterate the higher knock-out efficiency with\nHDCas12aCFD compared to HD_CFD, and hypothesize a novel role for the gene trade embargo\n(trem) in wing morphogenesis revealed by the superior efficacy of HDCas12aCFD.\nWhile initial impressions do suggest that HDCas12aCFD creates gene knock-outs with higher\nefficiency than Cas9 based HD_CFD, the authors need to address some major questions first,\nparticularly regarding extensive LOH, before they can fully establish that HDCas12aCFD is a\nsuperior gene knock-out tool.\nWe thank the reviewer for their thorough and detailed feedback. The reviewer raises an\nimportant concern: that clustered double-strand breaks (DSBs) could induce large deletions\nor chromosomal truncations, potentially explaining the strong phenotypes we observe with\nHD12aCFD. This concern is well-founded given extensive data from mammalian systems.\nHowever, whether such effects also occur with relevant frequencies in Drosophila has not\nbeen previously investigated.\nTo address this question, we performed multiple additional experiments that provide\nseveral new lines of evidence. These data demonstrate that copy-number neutral loss of\nheterozygosity (LOH) is the dominant mechanism in Drosophila, and that the enhanced\nphenotypes with multiplexed Cas12a mutagenesis are not caused by proximity effects.\nSpecifically, our new experiments show: (i) quantitative analysis of cells that gained or lost a\nLOH reporter reveals that copy-number neutral LOH predominates in flies; (ii) extended\nanalysis of genes neighboring those with known phenotypes does not support widespread\nproximity effects; (iii) phenotypes are not clustered along chromosome arms, with no bias\ntoward stronger phenotypes near centromeres, further arguing against proximity effects; and\n(iv) established differences in chromosome biology between Drosophila and mammalian cells\nprovide a mechanistic explanation for these observations. We detail these experiments and\nsupporting evidence below.\nThe major critiques are as follows:\n1) As stated in the opening paragraph, the ideal outcome for Cas9 or any other DNA DSB based\nmethod for gene knock-out is the generation of small indels around the targeted DNA site in any\nearly coding region. As long as the insertions or deletions do not generate 3 or multiples of 3 base\npair alterations, the gene will be knocked out of frame. More importantly, small indels ensure that the\ngenetic effect of DNA sequence modification is restricted to the target gene and the target gene\nalone, loss of larger amounts of DNA has the potential to affect neighbouring genes, which then calls\ninto question whether the phenotypic effect is from knocking out the targeted gene or any another\ngene that was also affected by a much larger deletion. The author’s extensive experiments with the\nLOH reporters unequivocally demonstrate that extensive LOH is a common and in some cases\nfrequent (~15-20%, Figure 4d) outcome of several HDCas12CFD arrays. While there is no further\ncharacterization of whether the LOH is a consequence of mitotic recombination (copy neutral) or\ndeletions/chromothripsis (copy loss), both outcomes are problematic for gene knockouts.\nWe have now analyzed whether the frequent LOH events we observe result from mitotic\nrecombination or deletion/chromothripsis. These mechanisms can be distinguished by their\nmolecular outcomes: mitotic recombination produces copy-number neutral LOH with\nbalanced proportions of cells gaining or losing a particular chromosome arm, whereas\ndeletions or chromothripsis result only in loss of genetic material without compensating\ngains.\nTo determine the relative contribution of each mechanism, we quantified the ratio of\nchromosome arm gain versus loss events using a heterozygous reporter gene. We performed\nthis analysis with five different HD12aCFD sgRNA arrays, two of which cut in close proximity\nto the reporter. The results, presented in the new Figure 5c and new Supplemental Figure 5,\ndemonstrate that cells with gained or lost reporter signals are present in approximately equal\nratios. This finding is consistent with mitotic recombination as the dominant form of LOH and\nargues against a significant role for extensive deletions affecting entire chromosome arms,\nas reported in mammalian systems (e.g., Lazar et al.).\nImportantly, established differences in chromosome biology provide a mechanistic\nbasis for the distinct behavior between Drosophila (and other Diptera) and mammalian\nsystems. Unlike mammalian cells, Drosophila somatic cells maintain homologous\nchromosome pairing in somatic cells throughout development (e.g. PMIDs 31582744,\n38722810), positioning homologs in close proximity and greatly increasing the likelihood of\ninter-homolog exchange following DNA double-strand breaks. We have incorporated this\nmechanistic explanation into the Discussion of the revised manuscript (Lines 600-607).\nHomologous recombination (HR) associated with crossing over could result in LOH, which could\nlead to a buildup of recessive alleles downstream of the recombination site. However, gene\nconversion associated with HR would also repair the broken DNA region on the targeted\nchromosome with DNA sequences from the homologous donor allele and thereby restore function,\nunless the donor allele was also mutated. This outcome would therefore suppress gene knock-outs.\nWe agree that repair of DSBs through homologous recombination is likely to restore the\noriginal sequence at the cut site. However, our system maintains Cas nuclease and sgRNA\nexpression for extended periods, enabling repeated targeting of restored sites.\nConsequently, cells that initially undergo copy-number neutral LOH have a high probability of\nsubsequent mutagenesis at the sgRNA target site, as repeated DSBs will eventually be\nrepaired through error-prone non-homologous end joining.\nWe also acknowledge that mitotic recombination leads to homozygosity of recessive\nalleles, potentially causing undesired phenotypes. However, experimentally induced LOH for\nmosaic analysis represents one of the most widely used genetic perturbation methods in\nDrosophila research (the foundational references PMIDs 8404527, 11311363, 1628809 have\nbeen collectively cited >3000 times), which has enabled numerous breakthrough discoveries\nin developmental biology and genetics. This extensive experimental precedent demonstrates\nthat in Drosophila, the risk from accumulating recessive mutations is manageable and that\nmethods inducing frequent copy-number neutral LOH are compatible with meaningful\nbiological investigations. The robust track record of LOH-based approaches in this model\norganism therefore supports the validity of our experimental strategy. We now discuss this\nimportant context in the revised manuscript (Lines 613-617).\nOn the other hand, deletions would lead to the same type of “proximity” effects reported by Lazar et\nal. (PMID: 38811841) for Cas9 based mutagenesis screens, as multiple genes will lose at least one\nallele downstream of the DSB if only one homologue arm was affected, and will be also knocked out\nif both homologues are affected.\nThe authors contend that LOH in drosophila occurs primarily through mitotic recombination.\nHowever, one of the two papers they cited, Brunner et al. (PMID: 31196871) characterized a random\nassortment of 57 events from a cohort of 253 recombination events induced by a Cas9 DSB, and\nreported that 19 contained an exchange of flanking DNA from both homologues without any indels,\n2 contained exchanges with indels, and the remaining 36 of 57 (~63%), which are a majority of these\n“recombination” events, were deletions that had lost regions of the chromosome distal to the DSB.\nThe cited experiment by Brunner et al. was performed on the 4th chromosome of Drosophila,\nwhich presents important limitations for comparison to our study. The 4th chromosome,\nwhich encodes only ~0.5% of protein-coding genes, is a specialized chromosome that lacks\nrecombination between homologs (PMIDs 28426351, 30401762), making it fundamentally\ndifferent from the major chromosome arms. Findings regarding recombination frequencies\non this chromosome therefore cannot be extrapolated to the bulk of the fly genome.\nAdditionally, the cited experiment showed a total recombination rate of only ~3% (253 of\n8,604 flies), therefore the absolute frequency of deletion alleles in this special szenario is\nbelow 2%.\nWe consider the study by Allen et al. (PMID 33444322) more relevant to our work, as\nthese authors analyzed multiple loci across the major chromosomes. Critically, this study\npresents extensive imaging data from imaginal discs following CRISPR-induced LOH\n(Figures 1, 2, 3, and 5), consistently showing approximately balanced levels of copy number\ngain and loss events. This pattern mirrors our results described above and strongly supports\nmitotic recombination, rather than deletions, as the predominant LOH mechanism.\nImportantly, Allen et al. used Cas9 rather than Cas12a, indicating that the balanced gain/loss\npattern we observe is not specific to Cas12a-mediated double-strand breaks but represents a\ngeneral feature of CRISPR-induced LOH in Drosophila somatic tissues.\nTherefore, the foremost concern regarding this study is that the primary source of the stronger\nphenotypic effects with HDCas12aCFD compared to HD_CFD is due to an increased incidence of\nextensive LOH that occurs via deletions, which then potentially nullifies the utility of this approach as\ngenotype to phenotype conclusions cannot be made if multiple genes are routinely knocked out at\nsignificant frequencies (~15-20%, Figure 4d). This possibility has not been addressed by the authors\nin any part of the manuscript. The authors performed Sanger sequencing around the DSB site for\nthree HDCas12aCFD arrays (0002, 0324 and 0476). As no local sequence effects were observed for\nthe low LOH score arrays 0324 and 0476, it may suggest DNA modifications at target sites by low\nefficiency arrays cannot be scored by Sanger sequencing. However, for the high LOH score array\n0002, sequence changes were observed around the DSB site, therefore local indel frequencies can\nbe estimated from algorithms such as TIDE (http://shinyapps.datacurators.nl/tide/) and ICE\n(https://ice.editco.bio/#/). And than this frequency could be compared to the % area of reporter loss\non wing discs as a measure of LOH frequency, to then compare frequencies of local deletions\nversus extensive LOH. And while it is true that even standard Cas9 based mutagenesis approaches\nlead to extensive LOH from deletions (references provided earlier), the study by Regan et al. (PMID:\n39386534) in mouse embryonic stem cells reported that for one of their extensively characterized\nCas9-gRNA sites, LOH comprised of 5% of all events, which would constitute a minor fraction of\nCas9 DSB repair outcomes compared to the ~85.5% that had indels as characterized by Sanger\nsequencing around the DSB site followed by analysis with ICE. This reviewer suggests the following\nanalyses to address the contribution of local sequence changes versus extensive LOH:\na) The authors have to perform more Sanger sequencing based analyses around the sgRNA array\nsites from a random assortment of their high LOH arrays and compare that to the absolute % of LOH\nthey observed in wing discs. They must then perform the same analysis with Cas9 arrays via\nHD_CFD, ideally around the same genetic loci, and only then can we evaluate if the increased knock\nout efficiency of the HDCas12a array is from an increase in local sequence changes versus\nextensive DNA loss. These experiments are strongly recommended.\nWhile we appreciate this experimental suggestion, we believe that correlating Sanger\nsequencing of PCR amplicons with LOH frequency has significant technical limitations that\nwould preclude definitive conclusions about large deletions and their impact on knockout\nefficiency. Most critically, extensive deletions at target sites cannot be detected by PCR\namplification, as deletion of primer binding sites prevents amplification entirely. Additionally,\nPCR exhibits inherent bias toward shorter products, causing local deletions to be\noverrepresented in sequencing data. These factors create a systematically biased view of on-\ntarget editing outcomes and severely limit quantitative assessment, particularly when\ndeletions are prevalent.\nOne might argue that samples showing detectable LOH but no apparent on-target\nmutations by Sanger sequencing (as presented in Supplementary Fig. 6) could indicate large\ndeletions. However, this interpretation overlooks a key mechanistic consideration: in\nsituations with limited cutting activity mitotic recombination might restore the original target\nsite without subsequent re-cutting and mutagenesis. Therefore, under these conditions the\ncombination of detectable LOH with undetectable on-target mutations is equally consistent\nwith copy-number neutral recombination.\nGiven these technical constraints, we believe the experimental approaches we\ndescribe in our revised manuscript provide more robust evidence to address the reviewer's\ncentral question: whether the enhanced phenotypes observed with HD12aCFD result from\nextensive deletions or reflect more efficient knockout of target genes. Importantly, while our\nexperiments tackle this question from different angles, they uniformly point to efficient target\ngene deletion rather than proximity effects as causative for the observed phenotypes.\nb) The authors assayed a number of sgRNA arrays for their effect on wing size and showed the\neffect of expected positive and negative controls met expectations. However, they also saw an effect\non wing size from a host of genes not known to affect this process, and there could be proximity\neffects in this dataset. Therefore, the authors should replot the data in Supplementary figure 7a,\nreorganizing the list of genes on the X-axis via their relative chromosome positions as opposed to\neffect size, and this could reveal if knocking out genes that are in the vicinity of and centromere\nproximal to genes that are known regulators of wing size also show proximity-based effects. This\nanalysis is also strongly recommended.\nThis has been an excellent suggestion, and we have now performed this analysis as shown in\nthe new Supplemental Figure 3. Using the karyoploteR R package, we accurately mapped\nwing size phenotypes obtained with different HD12aCFD sgRNA arrays relative to the\ngenomic position of their target gene. This analysis was facilitated by our extensive\nperturbation dataset (which we substantially expanded during the revisions and now\ncomprises 387 genes) and the compact organization of the Drosophila genome, with the vast\nmajority of genes located on just five major chromosome arms.\nIn contrast to human cells, where perturbation effects increase in strength when\ntargeting genes closer to centromeres (e.g., Figure 1 in Lazar et al.), we observe no such\npositional bias in our dataset. Genes causing wing size reduction are distributed randomly\nalong chromosome arms, with no enrichment near centromeres or other chromosomal\nlandmarks. This absence of proximity-dependent effects provides strong evidence that the\nmechanisms driving extensive deletions in human cells do not operate at significant\nfrequencies in the Drosophila system.\nc) Characterizing some of the LOH events, to determine if they are from reciprocal chromosome\nexchanges or deletions would also go a long way to alleviate the concern that large deletions are\nsignificantly affecting observed phenotypes. This analysis, however, is likely to be very intensive and\ntime-consuming, and may not even be feasible, even though these characterizations would be very\nconvincing one way or another.\nAs described above we have now carefully analysed the ratio of cells that have gained or lost\na chromosome arm and our finding that these cells occur at similar frequencies (new Fig. 5c),\nwhich is also supported by similar experiments by Allen et al. using Cas9, indicates that\nreciprocal chromosome exchange, and not large deletions, are the primary driver of LOH in\nour system.\n2) Another consequence of the occurrence of extensive LOH is they bring into question several\nsections of the paper. These are as follows:\na. The entire section entitled “Mutations caused by multiplexed editing are locally restricted” along\nwith figure 3 is rendered moot by the next sections that shows that extensive LOH is detected for\nmany sgRNA arrays. Moreover, if the results in Figure 3 are reevaluated after acknowledging that\nchromosomal sequences distal from the centromere and towards the telomere are often deleted\nfollowing DSBs induced with sgRNA arrays, it adequately explains why sgRNA arrays\ncentromere proximal to se at GstO3 affect its function but not at the telomere proximal Ra locus,\nwhen both arrays are practically equidistant from the beginning and end of se coding sequences\nrespectively and why the telomere proximal array at CG5892 has no effect on the centromere proximal\nebony locus. Internal sgRNAs at forked showed no effect, but this may reflect that forked is essential to\nviability in fruit flies and therefore large deletions are lost. Therefore, this entire section should be\nremoved, and could potentially be replaced with a further analysis of the deletion alleles generated\nat the target sites by sgRNA arrays used in actual gene knock-out studies, similar to the analysis in\nFigure 1C.\nWe respectfully disagree with this assessment. The experiments in Figure 3 (now revised\nFigure 4) directly address a critical practical question: whether our system generates false\npositives due to disruption of neighboring genes. We acknowledge that the original analysis\nof four gene pairs was limited in scope and have substantially expanded this investigation.\nWe selected additional gene pairs where the telomere-proximal gene has a well-\ncharacterized, strong knockout phenotype, while the centromere-proximal gene has an\nunknown phenotype. The revised Figure 4 now presents 10 additional comparisons, each\ninvolving a gene essential for wing morphogenesis. Targeting these reference genes with\nHD12aCFD sgRNA arrays produces strong wing size reductions, indicating highly efficient\nknockout. Critically, targeting closely neighboring genes does not affect wing\nmorphogenesis, demonstrating that proximity effects do not occur at practically relevant\nfrequencies.\nThese findings align with our previous data targeting genes near pigmentation loci,\nwhich revealed proximity effects for only one of four sgRNA arrays. Notably, this array\ntargets sites located towards the telomere and in extremely close proximity to sepia, located\njust tens to hundreds of base pairs from the transcriptional start site, a distance much closer\nthan the vast majority of target sites of HD12aCFD arrays are to neighboring loci.\nIn addition, we now have also investigated two HD12aCFD sgRNA arrays targeting\nnear haploinsufficient loci (new Suppl. Fig. 4). While rare in the genome, these loci are\nexpected to be particularly sensitive to proximity effects. We observed no phenotype in one\ncase and only mild phenotypes in the other, further supporting our conclusion that proximity\neffects are not a major driver of HD12aCFD phenotypes.\nCollectively, these experiments demonstrate that phenotypes typically result from on-\ntarget gene disruption rather than proximity effects. The results with GstO3 and one\nhaploinsufficient locus also illustrate that in rare cases neighboring loci can be affected,\ngiving rise to mild phenotypes, providing an important cautionary example that we now\nclearly highlight for users (Lines 609-617). The Drosophila community maintains rigorous\nquality control standards for genetic perturbation studies, including validation with\northogonal approaches, and is well-equipped to navigate such considerations.\nb. The section that utilizes LOH to determine off-target effects of sgRNA arrays along with\nexperiments in Figure 4 are unfortunately ill-conceived. The authors contend that a lack of off target\nLOH is evidence for lack of all off-target effects from sgRNA arrays. However, typical repair\noutcomes for Cas9 DSBs are mostly small indels, therefore off-target effects are also similarly\nexpected to mostly induce small indels. And it is important to acknowledge that the off-target sites of\nthe sgRNA arrays may not necessarily be clustered like the on-target site, therefore even if LOH\noutcomes are as frequent as localized indels on the sgRNA target sites, the same may not be true\nfor off-target sites. Off-target sites also may not be reproducibly cleaved, and thus the non-\nreproducible LOH observed could also be from off-target cleavage instead of spontaneous events.\nTo compare this to a recent study by Höijer et al. in Zebra fish (PMID: 35110541) that characterized\noff-target repair outcomes at predicted off-target sites, 76% were small deletions, 4% large deletions\n(>50bps) and 25% were small insertions. Even allowing for differences in repair outcomes between\nfruit flies and zebra fish and for Cas9 and Cas12a DSBs, nevertheless the LOH assay alone cannot\naccount for the presence or absence of small indels and therefore cannot eliminate the possibility of\noff-target effects. Unless the authors perform Illumina sequencing or other NGS on bulk DNA from\nflies that express a select few HDCas12aCFD arrays at multiple predicted off-target sites for all four\nindividual sgRNAs, the LOH analysis alone does not eliminate the possibility of off-target effects.\nWe agree with the reviewer regarding the importance of acknowledging limitations in our off-\ntarget analysis, and we have revised the manuscript accordingly (Lines 411-412, 625-628).\nLike any detection method, LOH screening has inherent sensitivity limits, and off-target\nevents occurring below this threshold could remain undetected. We now emphasize this\ncaveat more explicitly in the revised text.\nThe reviewer also raises an important mechanistic question that is important to judge the\nsuitability of our LOH assay for off-target detection: whether isolated DSBs caused by single\nsgRNAs (expected at potential off-target sites) also induce robust LOH . We have\nexperimentally addressed this question by directly comparing LOH induction between a\ncomplete HD12aCFD array and a single sgRNA targeting the same locus (for two independent\nloci). As demonstrated in the new Suppl. Fig. 7, single sgRNAs also induce robust copy-\nnumber neutral LOH, establishing that isolated DSBs at off-target sites would be detectable\nusing our screening approach. This result further supports the validity of our off-target\ndetection method, which we believe is one of the most innovative aspects of our study. We\nnote that systematic off-target data involving many sgRNAs has not been previously reported\nin Drosophila. Compared to our originally submitted manuscript, we have now doubled the\nproportion of the genome screened for nuclease activity (new Fig. 5j), substantially\nexpanding the data available to evaluate on- and off-target activity with HD12aCFD arrays.\nc. The final two sections entitled “Multiplexed Cas12a+ gene targeting outperforms established\nCas9-based methods” and “Increased gene disruption efficiency reveals a somatic function for trade\nembargo” are also complicated by the potential for LOH. Data in Figure 5 will be greatly\nstrengthened if LOH could also be assayed at any of the three genes targeted, if there is a\ngenetically assayable marker telomere proximal to these respective genes. And the novel function of\ntrem may also be a consequence of LOH mediated knock out of a gene telomere proximal to trem\non 3R, but this is a difficult question to address. Unfortunately, the two characterized mutants of trem\nwith identified indels both proved to be homozygous lethal, and thus the wing development function\ncould not be corroborated. Nevertheless, if the experiments previously suggested show LOH\nfrequency is comparable to local deletions for high efficiency sgRNAs, the assertions in Figure 5\nneed to be qualified by stating a lot of the cells with gene knock-outs potentially contain chromosome\ntruncations. And those in Figure 6 regarding a novel wing development function for trem ought to be\nremoved entirely, if the possibility of a confounding effect due to frequent LOH cannot be eliminated.\nThe experiments described above demonstrate that LOH in Drosophila is predominantly\ncopy-number neutral and that phenotypes from HD12aCFD gene targeting are typically not\ncaused by proximity effects. In addition, we have now obtained the two publicly available\nRNAi lines targeting trem and have analysed their effect on wing morphogenesis (see\nReviewer Figure below). One of those lines produces qualitatively identical, though\nquantitatively weaker, phenotypes to those observed with HD12aCFD arrays. Since RNAi acts\npost-transcriptionally without affecting genomic DNA, these phenotypes cannot result from\ndeletions or chromosomal rearrangements. This orthogonal validation strongly supports that\nthe observed phenotypes reflect genuine trem loss-of-function.\nThe trem experiments exemplify how the enhanced knockout efficiency of HD12aCFD arrays\ncan reveal previously undetected gene functions, demonstrating the biological value of this\napproach. We have therefore retained these findings in the manuscript as an important proof-\nof-concept for the system's discovery potential.\nTo reiterate, the major concerns regarding this manuscript involve experiments that address the\nfrequency of LOH and its potentially confounding effects on other assertions made in the manuscript,\nwithout which publication cannot be recommended. To summarize in a sentence, if the increase in\ngene knockouts from HDCas12aCFD is primarily due to an increase in extensive LOH, than this\nbasically nullifies the utility of this approach compared to conventional approaches that use one or\ntwo DSBs, instead of 4 clustered DSBs. Finally, a minor concern accompanying this is that\nmanuscript itself ought to be re-written, to acknowledge the potentially confounding affects LOH can\nhave for gene knock-out studies. Also, the reference in the legend for Figure 4 should refer to\nextended data Figure 3. Not 2.\nWe thank the reviewer for raising this critical concern. Given extensive data from other\norganisms, the hypothesis that multiplexed double-strand breaks could induce frequent large\ndeletions and proximity effects was indeed well-founded and merited thorough investigation.\nThe additional experiments we performed during revision have now definitively addressed\nthis possibility, demonstrating that induced LOH in our system is predominantly copy-\nnumber neutral rather than deletion-based and does not typically confound experimental\nresults. These findings reflect established differences in chromosome biology between\nDrosophila and other model systems. We have incorporated this mechanistic insight into the\nrevised manuscript to better contextualize our results within the broader literature.\nUltimately, the reviewer's feedback has helped us to substantially strengthen our manuscript\nand make it better accessible to the broad audience of Nature Communications.\nReviewer #2 (Remarks to the Author):\nIn this manuscript by Port et al., the authors describe a highly efficient gene-targeting system in\nDrosophila that leverages multiplexed Cas12a sgRNAs. By utilizing quadruple sgRNA arrays\nalongside an enhanced Cas12a variant (Cas12a+), they demonstrate the system's ability to\nefficiently mutate targeted genes. The authors suggest that this approach offers improved\nknockout efficiency compared to traditional Cas9-based methods, revealing phenotypes that previous\ntechniques may have missed.\nThe strength of this study is that it expands the CRISPR toolbox in Drosophila to Cas12a and\ndescribes effective strategies for constructing Cas12a/sgRNA reagents for tissue-specific gene\nediting. Using this strategy, the authors generated a small library of HD12aCFD sgRNA arrays\ntargeting 241 genes, which can be a useful resource for the Drosophila community. In addition, the\nauthors designed in vivo assays to test several other properties associated with CRISPR mutators\nthat have not been commonly examined in Drosophila, including DSB-induced toxicity, DNA\ndisruptions beyond the target sites, and off-target effects (even though that was limited to a specific\nchromosomal segment).\nWe thank the reviewer for their positive feedback. We are pleased to report that we have\nperformed a major extension of both our study and the associated transgenic resource\nduring revision. Our sgRNA library now encompasses lines targeting over 800 genes, which\nare now publicly available from the Vienna Drosophila Resource Center. We have also\ndoubled the scope of our off-target assessment and now report data covering 33% of the\nDrosophila genome.\nAlthough most of the experiments were nicely done and the results were mostly convincing (with the\ncaveat of missing some technical information that is important for evaluation), the reviewer thinks\nthat the authors have overstated the advantage of the Cas12a system over Cas9 systems. The\nauthors’ claims were based on unfair comparisons involving Cas9 sgRNAs with suboptimal designs\nand unequal numbers of sgRNAs for the two systems. Some caveats were presented as being\nunique to Cas9 systems but are in fact common to all CRISPR systems. Thus, the Cas12a system\nappears to be oversold while the strengths of the Cas9 system were understated.\nThe reviewer raises important questions about the relative advantages of the Cas12a-based\nsystem described here relative to currently used Cas9-based systems. In response, we have\nperformed extensive additional experiments, including two large-scale in vivo screens, and\nclarifications throughout the text. We believe these revisions fully address the reviewer's\nconcerns and provide a more complete assessment of our systems performance relative to\ncurrent technology. We have also carefully revised the manuscript to avoid overstatements\nabout the advantages of the HD12aCFD system. We detail our specific responses below.\nMajor concerns\n1. A major conclusion of this study was that the Cas12a+ and HD12aCFD system is superior to\nCas9 systems in gene mutagenesis (“gene targeting with Cas12a+ and multiplexed sgRNAs\nachieves superior gene disruption efficiency compared to established Cas9-based systems across\ndiverse tissues”.). However, this very general conclusion was based on incomplete/biased\ncomparisons. The efficiency of CRISPR mutagenesis is affected by multiple factors, including the\nchoice of targeting sites, gRNA scaffold sequence, the number of gRNAs used, and Cas protein\nexpression level and temporal patterns. In the comparison of Cas9 and Cas12a systems in this\nstudy, even if the Cas9 and Cas12a have comparable expression (of which the technical information\nis missing), the influence of gRNA target sites, numbers, and gRNA scaffold were not sufficiently\nconsidered. Numerous studies have shown that the gRNA target sequences have a large impact on\nediting efficiency (e.g. DOI: 10.1038/nmeth.3543; DOI: 10.1038/nbt.3437), but the manuscript did not\ndiscuss what criteria were used to select the target sequences for either system and what measures\nwere taken to ensure that they are efficient. More importantly, studies have shown that optimizing\nthe gRNA scaffold sequence for Cas9 can dramatically increase Cas9 activity (DOI:\n10.1016/j.cell.2013.12.001; DOI: 10.1126/science.aao0932). Further work in Drosophila has\nincorporated these optimizations and demonstrated their improved efficiencies over the original\nscaffold (DOI: 10.1534/genetics.118.301736; DOI: 10.1073/pnas.2014255118). However, the\nauthors have completely ignored these improvements in this manuscript, and the Cas9 sgRNAs\nexamined in this study seem to still use the original suboptimal scaffold. Thus, although the results in\nthis study suggest that the new HD12aCFD system is better than the authors’ own HD_CFD system,\nthey cannot be generalized for claiming that the Cas12a+ system is better than Cas9 systems. If this\nis the authors’ intention, they should compare HD12aCFD to similar Cas9 systems with the\noptimized gRNA scaffold and to make sure that the efficiency and the number of target sites for each\ngene tested are comparable for both systems.\nThis is a fair point. In vivo screens are time and labor intensive, so for practical reasons we\nhad initially focused our comparisons on the HD_CFD Cas9 library that was readily available\nin our lab. However, other relevant Cas9-based resources for gene knockout in Drosophila\ndiffer in construct design and could therefore have different knockout efficiencies.\nFurthermore, additional improvements to Cas9 sgRNA design have recently been described.\nThe reviewer is therefore correct to point out that the improved efficiency of the HD12aCFD\nsystem might be restricted to a subset of Cas9 systems. We have now addressed this\nlimitation through additional experiments.\nMost importantly, we have now compared our system to another major public\nresource for CRISPR knockout mutagenesis, which differs in important design parameters\nfrom the HD_CFD library. We ordered all available BDSC sgRNA lines targeting genes for\nwhich we also had HD12aCFD lines and performed side-by-side gene editing followed by\nquantitative assessment of wing size. The results presented in the new Figure 6g show that\nHD12aCFD lines identify phenotypes not recovered with lines obtained from the BDSC, but\nnot vice versa. This represents a major extension of our previous findings and demonstrates\nthat our newly described multiplexed sgRNA system also outperforms Cas9 systems with\ndifferent designs.\nWe agree with the reviewer that CRISPR mutagenesis efficiency can be strongly\ninfluenced by sgRNA design parameters, and that an ideal comparison would use matched\ndesigns. However, Cas12a and Cas9 use fundamentally different sgRNA scaffolds and cannot\nutilize sgRNAs with the same spacer sequences (due to different PAMs), preventing\nsystematic testing of matched designs. While this means individual comparisons might\nreflect variations in spacer efficiency, such differences would average out over many\ncomparisons. The fact that we performed over 250 pairwise comparisons across our two\nscreens and consistently observe cases where HD12aCFD arrays outperform Cas9 sgRNA\nlines, but not the reverse, strongly suggests a systematic advantage of this resource over\ncurrent technology. We agree it is important to clarify that these experiments inform which\ncurrently available resources are best suited for knockout mutagenesis, rather than which\nCas nuclease can in principle perform this task best. We have added a new paragraph to the\nDiscussion to make this distinction (Lines 568-581).\nWe also tested whether the improved Cas9 sgRNA vector suggested by the reviewer\n(described in PMID 33782117) could enhance the activity of existing sgRNA lines to match\nHD12aCFD arrays. Importantly, comparing two different Cas9 sgRNA vectors allows for a\nmatched design. We cloned the same sgRNA spacers used in the reference HD_CFD lines\ninto the improved pAc-U63-gRNA2.1 vector and inserted them into the same genomic landing\nsite. Any differences in activity must therefore result from the plasmid design rather than\ntarget site identity or expression level. We performed these comparisons on three central\nregulators of Hh, Wnt, and Notch signaling, genes for which the true loss of function\nphenotype is known. While sgRNA expression from the improved vector resulted in a mild\nincrease in phenotypic strength compared to the HD_CFD vector, HD12aCFD decisively\noutperformed both designs for all three target genes (new Suppl. Fig. 10).\nWe decided not to perform a comparison of Cas9 and Cas12a lines using the same\nnumber of sgRNAs in the arrays. While protocols for constructing larger Cas9 sgRNA arrays\nexist, they have a crucial practical disadvantage in that construction at scale is very cost and\nlabor intensive. A recent 4x Cas9 sgRNA library for the human genome required 168,000\nindividual oligonucleotides, over 125,000 individual PCRs, and over 40,000 GoldenGate\nreactions to construct 42,000 plasmids (see methods in PMID 39633028). The authors of this\nwork explicitly state that library construction took two full-time staff members 21 weeks. In\ncomparison, a library of similar size of 4x Cas12a sgRNAs can be generated using 42,000\npool-synthesized oligonucleotides (which are much cheaper than individual oligos) and a\nsingle large-scale PCR and GoldenGate reaction. A task that can be completed by a single\nresearcher in one week. Comparing 4x Cas12a and 4x Cas9 constructs would therefore\nsuggest a false equivalence between these approaches, when in reality one is practical to\ncreate at scale while the other is not. We have incorporated these points into the revised\nDiscussion.\nTogether, our new findings demonstrate that the multiplexed HD12aCFD sgRNA\narrays also outperform other Cas9 sgRNA libraries and designs. We regret not having\nprovided sufficient technical detail about the steps we took to ensure comparable sgRNA\nexpression and design (same landing sites, same sgRNA design software) in the original\nsubmission and have addressed this in the revised manuscript.\n2. The authors claimed that “Cas12a+ activity can be effectively controlled in time and space, as\nphenotypic changes remained confined to the targeted regions - a precision that has been\nchallenging to achieve with Cas9”. However, this point was not convincingly shown. The Cas12a+\nexpression in this study was controlled and determined by the Gal4 lines used, not by some\nintrinsic differences between Cas12+ and Cas9. Similar Gal4/UAS strategies can be used to control the\ntissue-specificity of Cas9 as well. To support this claim, the authors need to present data\ndemonstrating that Cas12a+ is intrinsically more specific than Cas9.\nWe agree with the reviewer that such a statement should be supported by more systematic\nand quantitative comparisons. As this is not a central point of our study we have opted to\nremove this section and plan to perform a comprehensive characterization of this parameter\nin the future.\n3. To show that gene disruptions caused by HD12aCFD sgRNA arrays are locally restricted, the\nauthors checked phenotypes associated with several recessive genes (Figure 3). However, to see\nthe recessive phenotypes, both alleles of the tested genes need to be mutated. Thus, the lack of a\nphenotype does not necessarily correlate with the lack of DNA disruption, and the results do not truly\nreflect the frequency of broader disruptions at the targeted loci.\nThank you for pointing this out. Haploinsufficient loci are rare in the Drosophila genome\n(PMID 22445104), so focusing on proximity effects for recessive genes represents the most\npractically relevant scenario. We have substantially expanded this analysis in the revised\nmanuscript (revised Fig. 4). However, we have now also analyzed proximity effects near two\nMinute loci, which encode haploinsufficient ribosomal protein genes (new Supplementary\nFig. 4). Disruption of just one copy of these genes is known to result in cells being lost\nthrough cell competition. We find no evidence for such effects when targeting one gene in\nproximity to a Minute locus, and only mild apoptosis when targeting the second. The latter\nobservation is consistent with disruption of neighboring loci at low frequency, although such\nphenotypes could also reflect fitness effects of the target gene itself, which remains\nuncharacterized. Overall, our expanded analysis of proximity effects shows that these are not\nmajor drivers of phenotypes observed with HD12aCFD arrays, but can occur in isolated\ninstances. We have taken care to present a nuanced discussion of these findings and their\nimplications in the revised manuscript.\n4. In somatic targeting of the STAR reporter, if the editing outcomes in the majority of the cells are\nfull deletions of the sequences between the first and last targeting sites, one would expect to see a\nrelatively distinct PCR band corresponding to such deletions (because shorter amplicons are\npreferred products in genomic PCR). However, Figure 1b does not show this. The results instead\nsuggest that full deletions are rare or at least low in frequency. The germline targeting results in\nFigure 1c cannot be used to infer somatic targeting, as DNA repair can differ significantly between\nthe soma and the germline. While I agree that the results suggest that deletions (that can be\nbetween any two target sites) become more frequent with more target sites overall, the writing\n(especially by describing the results of somatic and germline data in the same sentence) can leave\npeople a false impression about the frequency of large deletions in the soma.\nThank you for pointing this out. We have modified the text to provide a more nuanced\ndescription of our results. We now write that the smear observed in Fig. 1b indicates greater\ntarget locus diversification (resulting in more diverse heteroduplex amplicons). Deletions are\nlikely part of this diversification, but the reviewer is correct that it is not possible to make any\nstatements about their abundance from such gels and that differences in deletion\nfrequencies are likely to exist between different target loci and tissues.\n5. In Figure 1a, how would the editing outcome be different if only the most distal target sites were\navailable? In other words, how would the distance between two target sites affect the editing\noutcome?\nThat is an interesting question, but would require generating a new set of differently spaced\nSTAR reporters, which we consider to be beyond the scope of this manuscript. However, the\nspacing of target sites in endogenous genes is naturally variable and while there are more\nconfounding factors that complicate comparisons of mutation outcomes, the results\npresented in Fig 1d point towards increased target locus diversification also on these\nvariably spaced target sites.\n6. If the LOH events in the screening were truly due to background stochastic mitotic recombination,\nthey could be observable even in the control experiment where Cas12a or sgRNAs were not\nexpressed. Was this the case?\nWe have modified the text to provide a more balanced discussion of the three most likely\nexplanations for the rare and isolated LOH events: screening errors, background LOH and\nlow level off-target cutting. Obtaining definitive proof would be difficult for the first, require\nscreening of thousands of discs for the second, and high coverage trio whole genome\nsequencing for the last, and is therefore beyond the scope of this manuscript. We think it is\nbest to alert readers to all possibilities and in particular highlight that these could still be\ncaused by off-target effects that occur so rarely that they were not reproducible in the sample\nsize of our rescreen (now Line 411). While such off-targets would in most cases be too rare to\nsignificantly confound results, it is nevertheless important for users of this and similar\ntechnology to account for the possibility by using appropriate controls and validation\nstrategies.\n7. Some important technical information is missing for some experiments. It is difficult to evaluate the\nresults without the information. Below are some examples.\na. In Figures 1d, 3, and 5, where were Cas9 and Cas12a expressed? What measures were taken to\nensure that their expression levels were comparable? How were gRNAs expressed (by UAS or a\nubiquitous promotor)? What was done to ensure that the gRNAs were expressed at comparable\nlevels?\nb. In Figure 5, how was inducible mutagenesis of Notch, neur, and smo done?\nc. In Figure 1c, how many sequences are presented for each genotype/batch? How decisions were\nmade regarding which sequences are included in the figure panel?\nd. The manuscript did not talk about how gRNA target sites were selected and how to make sure\nthat they would be efficient.\ne. Scoring of the LOH index was not clearly defined.\nWe apologize for not always providing sufficient detail. We have now included these details\nin the figure legends and methods section of the revised manuscript and would like to thank\nthe reviewer for their feedback.\nMinor concerns\n1. “CRISPR editing often produces genetics mosaics”. This sentence reads as if genetic mosaicism\nis avoidable. But in tissue-specific CRISPR experiments, DNA DSBs are generated and repaired\nindependently in each cell in the manipulated tissue. The outcome should always be genetic\nmosaics.\nAgreed. We have revised this sentence.\n2. The authors claimed that “…for Cas9, the most widely used CRISPR nuclease, creating\nmultiplexed arrays of more than two sgRNAs remains technically challenging due to their large size\nand repetitive nature.” It’s true that gRNAs for Cas9 are larger than those for Cas12a, but\nmultiplexed Cas12a gRNAs also contain repetitive sequences. Several studies in recent years\n(including those published by some of the same authors) have already reported convenient methods\nfor making multiplexed (e.g. 2-6) sgRNAs for Cas9 (REF). So, I am not entirely convinced by the\nauthors’ argument that the Cas12a system is much better than Cas9 in this regard.\nThe crucial difference is that a quadruple Cas12a sgRNA array is small enough to be encoded\non a single synthetic oligonucleotide, while an array with the same number of Cas9 sgRNAs\nis too large. This allows Cas12a arrays to be produced and cloned in pools, whereas Cas9\nsgRNA arrays must be manually assembled. This makes cloning 4x Cas12a libraries orders\nof magnitude cheaper than 4x Cas9 sgRNA libraries. Please also see our response above,\nwhich illustrates this contrast with a concrete example.\n3. Not all results in Supplementary Fig. 5 were described in the main text. Although some details\nwere provided in the figure legend, the results should be at least briefly described in the main text.\nThank you for pointing this out. This has been corrected.\n4. Supplementary tables were not provided.\nThere appears to have been a problem during the upload of the initial submission. We now\nprovide all tables.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nPort et.al. present a Cas12a-based multiplexed system that targets each gene with four sgRNAs to\nenhance editing efficiency, using Drosophila as an in vivo model. Their findings demonstrate that the\nsynergistic action of multiple sgRNAs leads to efficient deletion formation, significantly increasing the\nproportion of loss-of-function mutations. To rigorously evaluate off-target effects, they introduce a\nnovel screening assay that enables real-time visualization of CRISPR-induced chromosomal\nalterations in living organisms and demonstrated no detectable off-target effects. Furthermore, they\nreveal the key roles of trem in wing development by using multiplexed Cas12a system. Overall, the\nstudy is intriguing and most of the data support their conclusion. My major concern is that the\nCas12a multiplexed system in Drosophila has been described by the same group but there is no\nclear description that what the improvement of the system is and there is no data to support this\nsystem is outperform than previous one.\nWe would like to thank the reviewer for their valuable feedback. Our 2020 paper (PMID\n32843348) represents a proof-of-principle study that shows that Cas12a+ can in principle be\nused for multiplexed gene editing in Drosophila. Many highly influential method papers were\nnot the first ones describing an approach, but rather those that made it practically usable\n(e.g. Brand and Perrimon, 1993 (>9000 citations) or Bischof et al., 2007 (>1900 citations)).\nThese studies rigorously evaluated the potential of a previously described technology and\nintroduced refined tools to use it. Our current manuscript follows this tradition by providing\nnovel mechanistic insights into the mode of action of multiplexed gene targeting,\nsystematically evaluates its safety and efficiency, and introduces a large-scale sgRNA\nresource accessible to the community through the Vienna Drosophila Stock Center. Such\ndata and resources had so far been missing and represent a requirement for broad\ncommunity adoption.\nIn addition, we are convinced that the use of LOH screening to visualize CRISPR\nactivity over entire chromosome arms is an innovation of broad interest to the genome\nediting community. We are not aware of previous work describing the unbiased assessment\nof CRISPR activity using thousands of sgRNAs over a very large proportion of the genome.\nGiven that off-target propensity of sgRNAs is highly variable and bioinformatic prediction of\noff-target sites imperfect, the scale of the search space and sgRNA number that can be tested\nwith this technology are key advantages that will be of broad interest to the community.\nSpecific comments:\n1.Figure 1a illustrates the report structure targeted by the Cas12a system, where the target sites are\nidentical. However, there is no description of the structure of the multiplexed sgRNAs array. Are the\nsame sgRNAs used to target these identical sites, or is the array multiplexed with various sgRNAs?\nIncluding a diagram to depict the Cas12a sgRNAs system would be helpful. Additionally, if the array\nreporter could be designed to contain different target sites and be targeted by six distinct sgRNAs\n(with similar editing efficacy), it would provide further insights into assessing editing efficacy.\nThe reporter described in Fig. 1a-c uses multiplexing of the same target site that is targeted\nby a single sgRNA. The advantage of this system is that each site is targeted with the same\nprobability, which allows us to assess how mutagenesis patterns evolve if additional cut\nsites are added. When targeting endogenous genes the efficacy of individual sgRNAs and\ntheir spacing is highly variable, which substantially complicates such analysis. We directly\nshow this complexity in Figure 1d, which we believe is more relevant than generating a\nreporter with diverse cut sites. We have revised the text to more clearly describe the reporter\nsystem and the rationale behind it.\n2.What is the x-axis in Figure 1c? How can the non-linear changes in deletion percentage among\narrays be explained? For instance, why does the 2x array show a higher percentage of deletion than\nthe 3x array in the first region? Additionally, while the 4x array exhibits the highest deletion\npercentage, there is a decline in the 5x and 6x arrays. Clarification on these trends would be helpful.\nFigure 1c shows schematics of different germline transmitted alleles of the STAR reporter.\nThe X axis represents the sequence of the reporter. Each line represents a sequencing read\nfrom an animal that has been individually genotyped, a laborious process that limits\nthroughput of this assay. We believe this data clearly shows the potential to induce deletions\nbetween multiplexed target sites and also the heterogeneity of outcomes that stems from the\nfact the different repair modes compete with each other. However, we think to quantitatively\ncompare the relative abundance of different outcomes between different designs would\nrequire many more datapoints, which would be practically difficult to achieve. We have edited\nthe text and figure to clarify these points.\n3.In Figure 1d, the authors compared the Cas12a multiplexed system with the Cas9 system. To\ndraw a solid conclusion that Cas12a outperforms Cas9, it would be helpful to include the deletion\nefficacy of each single guide used in Figure 1d for both Cas12a and Cas9. As well as showing the\ndeletion efficacy of Cas12a with two guides. Also, in figure 5 and 6.\nWhile in principle such comparisons can be performed, as showcased in Fig. 1c, they are\nextremely laborious when performed over many conditions at the appropriate scale. As the\nmutation profile alone does not inform about the efficiency of gene knock-out (as multiple\npotential mechanisms of biological compensation exist), we have instead focused our efforts\non answering a related question that is of higher practical relevance: Does multiplexed\nCas12a gene targeting result in efficient and specific ablation of gene function? We believe\nour revised manuscript clearly shows that this is the case.\n4.Although, the claim ‘the use of multiple sgRNAs creates functional redundancy’ has been\ndemonstrated by previous studies, the data in Figure 1 does not provide enough support for this\nclaim. The authors could strengthen this point by citing relevant literature or including additional\ndata, such as single sgRNAs efficacy controls, to better support their argument.\nWe have revised the text and included additional citations to better support this argument.\n5.Please explain the reason that the mutation test in figure 3 only use up to 2 guides instead of 4\nguides.\nWe have performed a major extension of these experiments and present them now in the\ncompletely revised Figure 4. All of the described experiments used HD12aCFD arrays, which\nencode 4 sgRNA per gene.\n6.The authors claim that off-target effects of Cas12a editing are undetectable. Is this due to the\nlimitations of the detection system? Whole-genome sequencing would be useful to determine\nwhether low-frequency off-target effects are present. Does these low off-target effects are universal\nin high order model or only in fly model. For future applications, in vivo off-targets information in\norganoid or mice model are useful (organoid or mice).\nLike any other off-target detection method LOH screening will have a detection limit, which is\nset by the ratio of LOH and mutations at the cut site and the number of cells that are analysed\nin a given experiment. The data we present in Suppl. Fig. 6 shows that the sensitivity of the\nassay makes it well suited to detect off-targets that occur with practically relevant\nfrequencies (it is important to note that what constitutes such ‘practically relevant\nfrequencies’ will be very different when considering research in a model organism (the focus\nof this manuscript) or for example medical applications of CRISPR technology). While whole-\ngenome sequencing is indeed a powerful way to detect off-targets, it is technically very\nchallenging to distinguish natural genetic variation from off-target mutations in WGS data. As\na result, informative experiments of that kind are rare and constitute entire manuscripts on\ntheir own. Moreover, WGS in combination with many sgRNAs, a crucial factor given the\nheterogeneity of off-target potential, is cost prohibitive. We therefore consider the use of\nWGS beyond the scope of this manuscript. While performing off-target analysis in mice or\norganoids is not the topic of our current study, we do provide references that highlight that\nLOH screening is also feasible in mammalian models.\n7.Statistics is missing in figures 6b, 6c, and 6d.\nThank you for highlighting this omission, which we have now corrected.\nNCOMMS-25-02242A: Point-by-point response – final revisions\n[Author responses are in bold]\nReviewer #1 (Remarks to the Author):\nFollowing on from the initial review of the manuscript, the primary concern raised by this reviewer\nwas the possibility of chromosome-arm scale deletions induced by clustered Cas12a DSBs\nleading to copy number-loss Loss of Heterozygosity (CL-LOH), which would produce\nconfounding proximity based phenotypic effects and limit the efficacy of this approach as a novel\ngene knockout strategy. Upon reading the revised manuscript, this reviewer commends the\nauthors for embracing this concern and going above and beyond to address it. The experiments\nin the revised manuscript thoroughly demonstrating an absence of proximity based phenotypic\neffects from gene knockouts, and the presence of copy number gain events strongly support a\npredominance of DSB induced CN-LOH and a relative absence of DSB induced CL-LOH events.\nMoreover, the authors also produced a supporting figure showing that RNAi mediated trem\nknockdown also shows a similar, albeit weaker wing phenotype as the HDCas12aCFD mediated\ntrem knockout, which ameliorate concerns regarding the novel proposed function of trem. This\nreviewer therefore recommends publication, notwithstanding a few minor comments that can be\naddressed with a few small changes in the manuscript text, which are as follows:\nWe would like to thank the reviewer for their kind words and their constructive and helpful\nfeedback throughout the review process.\n1) The copy-number neutral LOH (CN-LOH) events in the paper are ubiquitously described as\nmitotic recombination events. While it is true that allele specific Cas9 DSBs induce substantial\nlevels of mitotic recombination associated with crossing over in Drosophila (PMID: 33750946), an\nimportant difference is that DSBs induced in this paper produce breaks on both homologues.\nThis raises the formal possibility that CN-LOH events instead occur via reciprocal end-joining\nevents, which have been demonstrated in human HT1080 cells, a p53-positive fibrosarcoma cell\nline (PMID: 32873648). The reviewer does acknowledge that frequent mitotic recombination has\nbeen demonstrated before in Drosophila, nevertheless acknowledging the possibility of reciprocal\nend-joining events covers all bases.\nThank you for this excellent point. We have adopted the broader term “interhomolog\nrecombination” in lines 260, 453, 460 and now mention the possibility of reciprocal end\njoining in line 452 and the legend of Fig. 5a.\n2) The method of tracking LOH on chromosome arms with integrated fluorescent reporters to\ndetect off-target genome editing is indeed very convenient, but this approach is limited to tracking\nevents only on the marked chromosome arms. This caveat is usually not acknowledged in the\ntext, rather the phrase “off-target activity” is used without any qualifiers. It is therefore\nrecommended that the text in lines 91 and 420 be changed to “off-target activity on the two\nchromosome arms with LOH reporters”.\nWe qualify the scope of our off-target assessment in lines 291, 292 (“any array-encoded\nsgRNAs with off-target activity on the screened chromosome arm are expected to lead to\nreproducible LOH”), lines 302, 303 (“In summary, LOH visualization enabled us to screen\nfor Cas12a+ activity with over 2000 sgRNAs across 33% of the Drosophila genome.), and\nlines 470-472 (“Screening for nuclease activity across 33% of the Drosophila genome in\ncombination with over 2000 sgRNAs failed to detect any reproducible cases of off-target\nactivity.”).\n3) Figure 3 legend line 244 refers to supplementary figure 1, when it seems it should refer to\nsupplementary figure 2.\nThank you. This has now been corrected.\nReviewer #2 (Remarks to the Author):\nThe authors have done a very nice job revising the manuscript by adding substantial new results.\nThese new results and revisions of text addressed my previous concerns. The revised\nmanuscript is a very nice piece of work which represents a significant advancement in CRISPR\ntechnology and application in Drosophila. I am supportive of its publication by Nature\nCommunications but with two minor comments.\nWe would like to thank the reviewer for their encouragement and valuable feedback that\nhas led to a significant improvement of our study.\n1. Figure 2 characterized Cas12a+ activity using the GRACE reporter. UAS-Cas12a+ appears to\nhave low levels of leaky activity in the wing disc. How is it compared to the leaky activities of\nprevious UAS-Cas9 strains generated by the same group? This information would be useful for\npeople deciding between the Cas9 and Cas12a+ system.\nWe appreciate this comment. While we have anecdotal observations suggesting that\nCas12a+ exhibits reduced leaky activity compared to some of our previous Cas9 lines, a\nrigorous comparison would require testing multiple target sites, as differences in\nbackground activity could reflect variations in sgRNA efficiency rather than inherent\ndifferences between nucleases. We plan to address this in future work.\nThe current manuscript focuses on demonstrating the substantial improvements in\nknockout efficiency achieved with the HD12aCFD system and establishing the high\nspecificity of Cas12a+-mediated mutagenesis.\n2. It is not clear how the sequencing results in Supplementary Figure 6 were generated. Were\nthey derived from PCR from a single animal or a mixture of multiple larvae? Or were they from\ndissected larval wing discs?\nWe have clarified the experimental procedure in the figure legend of Suppl. Fig.6.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed most of my comments. I don't have any further questions.\nThank you for your valuable feedback throughout the review process."}]

Decision letter