The impact of genome editing on the introduction of monogenic traits in livestock

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1 Genetics Selection Evolution RESEARCH ARTICLE Open Access The impct of genome editing on the introduction of monogenic trits in livestock John W. M. Bstinsen *, Henk Bovenhuis, Mrtien A. M. Groenen, Hendrik Jn Megens nd Hn A. Mulder Abstrct Bckground: Genome editing technologies provide new tools for genetic improvement nd hve the potentil to become the next gme chnger in niml nd plnt breeding. The im of this study ws to investigte how genome editing in combintion with genomic selection cn ccelerte the introduction of monogenic trit in livestock popultion s compred to genomic selection lone. Methods: A breeding popultion ws simulted under genomic selection for polygenic trit. After reching Bulmer equilibrium, the selection objective ws to increse the llele frequency of monogenic trit, with or without genome editing, in ddition to improving the polygenic trit. Scenrios were compred for time to fixtion of the desired llele, selection response for the polygenic trit, nd level of inbreeding. The costs, in terms of number of editing procedures, were compred to the benefits of hving more nimls with the desired phenotype of the monogenic trit. Effects of reduced editing efficiency were investigted. Results: In popultion of 20,000 selection cndidtes per genertion, the totl number of edited zygotes needed to rech fixtion of the desired llele ws 22,118, 7072, or 3912 with, no, moderte, or high selection emphsis on the monogenic trit, respectively. Genome editing resulted in up to four-fold fster fixtion of the desired llele when efficiency ws 100%, while the loss in long-term selection response for the polygenic trit ws up to seven-fold less compred to genomic selection lone. With moderte selection emphsis on the monogenic trit, introduction of genome editing led to four-fold reduction in the totl number of nimls showing the undesired phenotype before fixtion. However, with currently relistic editing efficiency of 4%, the number of required editing procedures incresed by 72% nd loss in selection response incresed eight-fold compred to 100% efficiency. With low efficiency, loss in selection response ws 29% more compred to genomic selection lone. Conclusions: Genome editing strongly decresed the time to fixtion for desired llele compred to genomic selection lone. Reduced editing efficiency hd mjor impct on the number of editing procedures nd on the loss in selection response. In ddition to ethicl nd welfre considertions of genome editing, creful ssessment of its technicl costs nd benefits is required. Bckground Animl breeders hve long history in chnging the genetic mkeup of livestock. Until recently, this hs only been possible through trditionl selective breeding, which is reltively slow process tht ccumultes desired lleles over mny genertions. In the 1980s nd 1990s, the possibility of developing trnsgenic livestock seemed to offer n lterntive pproch of *Correspondence: john.bstinsen@wur.nl Wgeningen University & Reserch Animl Breeding nd Genomics, PO Box 338, 6700 AH Wgeningen, The Netherlnds chieving selection response. By pplying trnsgenic methodologies, gene could be inserted into the genome of n orgnism t rndom positions, but this pproch ws hmpered by technicl difficulties nd limittions nd rised public concerns. So fr, no trnsgenic livestock hve been pproved for humn consumption, except trnsgenic slmon in the USA [1, 2]. The dvent of genome editing (GE) hs dded new possibilities for ltering genomes. New technologies such s CRISPR-Cs9 hve considerbly improved the level of efficiency nd precision of modifying the genome The Author(s) This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( publicdomin/zero/1.0/) pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Pge 2 of 14 compred to the trnsgenic methodology. Editing cn be guided to ny specific loction in the genome nd could be used to chnge genes so tht they produce different product or become non-functionl genes. It hs been suggested tht CRISPR-Cs9 cn be used to repir genetic defects, s demonstrted in mice [3], or to confer resistnce to diseses, s reported in whet nd rice [4]. Progress in GE hs lso been mde in cttle, sheep, pigs nd gots [5 8], for instnce to confer resistnce to diseses [9] or introduce polledness (bsence of horns) [10]. GE techniques hold much promise for the genetic improvement of livestock nd hve the potentil to become the next gme chnger in niml nd plnt breeding. Genome editing in livestock hs primrily introduced lleles tht ffect monogenic trits [9, 10]. Improving polygenic trit by promotion of lleles by genome editing (PAGE) in combintion with genomic selection ws simulted by Jenko et l. [11] nd shown to increse response to selection fter 20 genertions by to 4.12-fold compred to using genomic selection lone. In their simultion, it ws ssumed tht genes nd their effects were known without error. However, one of the mjor technicl obstcles for implementtion of GE in commercil breeding progrms is the limited knowledge regrding the custive muttions tht underlie the observed genetic vrition. Therefore, PAGE my not be of interest for quntittive nd complex trits in the ner future becuse editing trgets re lcking. Alterntive pproches hve been suggested tht pply editing to mny loci to simultneously prove nd use the effect of vrints [12]. Designs for these schemes need to be developed nd tested. Introduction of lleles by GE my hve severl dvntges compred to clssicl breeding strtegies including genomic-ssisted introgression. Clssicl introgression breeding strtegies re difficult, costly nd time-consuming, nd lso suffer from lower genetic gin, linkge drg nd incresed inbreeding in the region surrounding the trget gene [13, 14]. The use of genomic dt cn improve the efficiency of removing the donor genome [13 16] nd further increse genetic gin [17, 18], but it hs not led to the ppliction of introgression in livestock. When monogenic trit is lredy present in the popultion, incresing the frequency of the desired llele from low strting vlues with clssicl breeding strtegies is still difficult nd results in incresed inbreeding nd decresed genetic gin in the totl breeding gol [18, 19]. An exmple is the polled llele tht is desired to breed diry cttle without horns. Currently, horns re removed to prevent cttle from hurting ech other or humns. However, dehorning is n invsive nd pinful procedure, which is expected to become further regulted or bnned in some countries. In cttle, single gene is responsible for polledness, but the corresponding llele is very rre in diry cttle. It my esily tke 20 to 30 yers to rech fixtion, nd therefore this gene is possible trget for GE, since it would not hve the disdvntges observed with selection provided tht enough nimls re edited successfully [11]. To dte, it is not known how genomic selection nd GE could be combined to reduce the number of nimls to be edited, increse the llele frequency of desired llele, nd minimize the loss in genetic gin for other trits. Furthermore, the current efficiency of GE is low nd the mortlity of genome-edited zygotes is high [20]. Although CRISPR-Cs9 is much more precise technique for GE thn previous genetic modifiction techniques, edited nimls my show off-trget effects [21], which would result in culling selection cndidtes. The im of this study ws to investigte the extent to which GE, in combintion with genomic selection, could contribute to the chnge in frequency of n llele with monogenic effect compred to genomic selection lone. We investigted the effects of the weight on the desired llele in the breeding gol, the rte of success of GE, nd the survivl rte of edited zygotes on the chnge in llele frequency using Monte Crlo simultion. Furthermore, the effects on genetic gin in the polygenic trit nd on the rte of inbreeding were ssessed. The benefits from hving nimls with the desired trit nd the cost, in terms of number of nimls to be edited, were compred. Methods The im of this study ws to mimic livestock breeding popultion tht, historiclly, hd been selected for polygenic trit (or index of trits). Strting in genertion 0, incresing the frequency of the desired llele of monogenic trit, such s polledness in cttle [22, 23], ws dded to the breeding gol. Simultion of nimls A breeding popultion ws simulted with discrete genertions consisting of 100 mle prents nd 2000 femle prents per genertion (Fig. 1). To produce the next genertion, one mle prent ws rndomly ssigned to ech femle prent. Ech mting produced 10 offspring, resulting in 20,000 nimls per genertion with rndom sexes ssigned with 0.5:0.5 probbilities. Animls in the founder genertion received true breeding vlue (TBV) for the polygenic trit by drwing rndom vlues from norml distribution with men of zero nd vrince of 1 (N(0,1)) using the function rnorm()in R softwre [24]. TBV of the offspring in lter genertions were hlf the TBV of the sire plus hlf the TBV of the dm plus Mendelin smpling effect drwn from norml distribution

3 Pge 3 of 14 Fig. 1 Schemtic representtion of the simulted breeding progrm N(0, 0.5(1 0.5(F sire + F dm ))). A genotype for the monogenic trit ws ssigned to ll founders bsed on Hrdy Weinberg equilibrium proportions nd frequency for the desired llele of A low strting frequency ws ssumed to llow comprison to selection scenrios without GE. The monogenic trit ws controlled by single gene with no effect on the polygenic trit, such s the dominnt trit polledness in cttle [22, 23] or the recessive trit resistnce to Escherichi coli F18 in pigs [25]. For these exmples, the strting frequency of the desired llele would be higher thn the 0.01 vlue tht ws chosen s the strting point for simultions. In ddition to the min scenrio with 100 mles, 2000 femles nd 10 offspring per mting, dditionl scenrios were simulted with popultion sizes tht were similr to rel breeding progrms in diry cttle, pigs, nd fish. In diry cttle, the numbers of mles nd femles per genertion nd the number of offspring per femle were 200, 600, nd 16, respectively. These numbers were 50, 2000, nd 20, nd 120, 240 nd 40, for pigs nd fish, respectively. A smll breeding progrm ws lso included with 20 mles, 240 femles, nd 70 offspring per femle. Other prmeters for these species-specific designs were kept the sme s in the min simultion scenrio. Selection Selection ws simulted in two phses. The first five genertions of selection were only on the estimted breeding vlue (EBV) of the polygenic trit. This first phse ws used to rech equilibrium genetic gin, fter reduction of the genetic vrince due to the Bulmer effect [26]. Trunction selection ws pplied to choose 100 sires nd 2000 dms with the highest EBV from the totl popultion of 20,000 cndidtes. The defult breeding progrm, without GE, ws scheme where selection ws for genomic EBV with relibility of 0.5 (r 2 ). The EBV ws simulted by dding to the TBV prediction error (PE) tht ws drwn from norml distribution, N(0, 1 r2 ), i.e. r 2 EBV = TBV + PE. Subsequently, the EBV were scled to the proper vrince by multiplying them with r 2. In the second phse, the sme numbers of sires (100) nd dms (2000) were selected from 20,000 cndidtes in ech of the 20 genertions by trunction selection on n index, I = b 1 EBV + b 2 G, where b 1 nd b 2 were the index weights nd G ws the number of desired lleles for the monogenic trit. The EBV ws clculted s in phse 1 nd the genotypes for the monogenic trit were ssumed known without error. Index weight b 1 ws fixed to 1 nd b 2 hd vlue of 0, 0.5, or 1000 (Tble 1). With b 2 = 0, selection ws only on the EBV, i.e. genomic selection on the polygenic trit. With b 2 = 1000, mximum emphsis ws put on the monogenic genotype. The vlue of b 2 = 0.5 ws chosen empiriclly so tht the chnge in frequency of the desired llele followed n intermedite pttern. Genome editing We ssumed tht GE of the gene tht ffects the monogenic trit ws pplied to zygotes during reproduction.

4 Pge 4 of 14 Tble 1 Scenrios simulted Method b 2 k b s c N d edited GS + GE , 0.2, 0.4, 0.6, 0.8, 0.05, 0.2, 0.4, 0.6, , Index weight for the monogenic trit genotype (I = b 1 EBV + b 2 G), sme b 2 vlues were used for GS b Probbility of successful editing c Probbility of survivl for edited offspring d Number of zygotes edited After mte ssignment, mtings were selected for editing nd the resulting zygotes were subjected to GE procedures nd could led to edited offspring. The genotype of the selected prents did not chnge. In the different scenrios, GE ws pplied to the zygotes of either none of the mtings, 10% of the mtings, or ll mtings. When editing ws limited to 10% of the mtings, we selected those tht (1) hd the smllest number of desired lleles for the monogenic trit in the prents nd (2) the best prent verge for I. Genome editing chnged ll undesired lleles in the offspring into the desired lleles. An editing success probbility, k, ws pplied seprtely to ech llele nd ws set to 1.00, 0.80, 0.60, 0.40, 0.20 or 0.10 in different scenrios (Tble 1). A probbility of surviving the editing procedure, s, ws pplied to ech zygote tht ws subjected to editing nd ws set to 1.00, 0.80, 0.60, 0.40, 0.20 or 0.05 in different scenrios (Tble 1). Editing success nd editing survivl were the result of Bernoulli tril with, respectively, probbilities k or s. Vlues of s lower thn 1.00 resulted in edited fmilies hving fewer offspring thn fmilies tht were not selected for GE. Currently, efficiency of GE is low, with one live edited offspring for 24 edited zygotes reported in the literture [20]. To mimic this low efficiency, n editing success probbility k of 0.20 ws combined with survivl probbility s of 0.20 in order to hve one live edited offspring for every 25 zygotes edited. These vlues were used in GS + GE scenrio with b 2 = 0.5. Evlution of scenrios All scenrios were replicted 50 times. The number of editing procedures ws counted t ech genertion s the number of zygotes tht were genome-edited. Genome editing ws pplied only to fmilies in which the prents crried t lest one copy of the undesired llele. The genetic level in ech genertion ws clculted s the verge TBV nd selection response ws clculted s the difference in verge TBV between the current nd previous genertion. Inbreeding of ll individuls ws clculted with the function clcinbreeding() of the pckge pedigree [27] in R [24]. The inbreeding level in ech genertion ws clculted s the verge inbreeding coefficient, nd inbreeding rte ws clculted s the difference in verge inbreeding coefficient between the current nd previous genertion. Frequency of the monogenic llele nd the vrince of TBV were clculted per genertion. Compring costs nd benefits Choosing GE to increse the frequency of desired llele will depend on, mong other considertions, the costs nd benefits of lterntive pproches. The cost benefit comprison ws performed t breeding horizon of 5 genertions nd t 20 genertions. Costs of using GE were ssumed to be minly due to the editing procedures. The cumultive number of edited zygotes ws used s mesure of costs. Benefits of using GE were clculted s the extr offspring tht hd the desired phenotype due to ppliction of GE. Between scenrios, we compred the percentge of nimls with the desired phenotype for the monogenic trit, ssuming either dominnt or recessive gene ction. Adding the monogenic trit to the index will increse the frequency of the desired llele but, t the sme time, reduce selection response for the polygenic trit. The prmeters ssessed in genertions 5 nd 20 were the loss in selection response for the polygenic trit in genetic stndrd devitions nd in months, ssuming genertion intervl of 2 yers, the cumultive inbreeding, nd the frequency of the desired llele. The loss in selection response ws bsed on the difference in men TBV of specific scenrio nd the men TBV in the sme genertion in the GS scenrio with b 2 = 0. The loss in selection response is presented in months by dividing this difference in men TBV by the equilibrium response in genertion 0 nd multiplying by the genertion intervl of 24 months. The loss in selection response is lso presented in genetic stndrd devitions using equilibrium genetic vrince in genertion 0. Results After five genertions of selection on only the EBV for the polygenic trit in phse 1, the equilibrium genetic vrince (σ A 2 ) nd equilibrium selection response were reched with genetic vrince of 0.73 nd selection response per genertion of The llele frequency of the monogenic trit in genertion 0 remined t the strting frequency, on verge 0.01 ± The desired llele of the monogenic trit ws lost due to drift in 14 out of the 950 replictes cross ll scenrios (1.5%).

5 Pge 5 of 14 Frequency of the monogenic llele The time to fixtion for the monogenic genotype ws very short with the mximum weight on the monogenic genotype nd very similr to tht obtined with four genertions by GS nd three genertions by GS + GE (Tble 2; Fig. 2). With moderte weight on the desired llele (b 2 = 0.5), the time to fixtion becme very different between scenrios with 17 genertions for GS nd only five genertions for GS + GE (Tble 2). With moderte weight on the desired llele, GE gretly reduced the time to fixtion. In most scenrios, the desired llele becme fixed (Fig. 2), with few exceptions. The llele frequency did not chnge in the GS scenrio with zero weight on the desired llele (b 2 = 0). In the GS scenrio with b 2 > 0, the desired llele ws fixed in ll except three of the 100 replictes for which the desired llele ws lost due to drift (Fig. 2). The GS + GE scenrios lwys resulted in fixtion of the desired llele becuse even if the desired llele ws lost due to drift, it ws re-introduced by GE. With zero weight, fixtion ws reched fter 13 genertions. Selection response for the polygenic trit With b 2 = 0, selection in phse 2 remined the sme s in phse 1: genomic selection for the polygenic trit. Due to inbreeding, the selection response for the polygenic trit decresed from n equilibrium response of 1.13 in genertion 0, to 1.09 in genertion 20 (Fig. 2). When mximum emphsis ws given to the desired llele (b 2 = 1000), shrp decrese in the response for the polygenic trit ws observed in genertions 1, 2 nd 3 (Fig. 2). In genertion 2, the response ws only 29% of the equilibrium selection response in genertion 0. When combined with GE, shrp decrese in selection response ws still observed, but only in genertions 1 nd 2 (Fig. 2), nd the lowest response still reched 62% of the equilibrium response in genertion 0. Thus, loss in polygenic response due to selection for the monogenic trit ws more thn hlved by GE. With moderte weight on the desired llele (b 2 = 0.5), the reduced response lsted much longer, until genertion 9, for the GS scenrio (Fig. 2). In the GS + GE Tble 2 Genertions to fixtion of monogenic trit b 2 GS b 0 inf d d 3 GS + GE c Index weight for the genotype of the monogenic trit b Genomic selection only c Genomic selection + genome editing d Time to fixtion in replictes in which the desired llele is not lost due to drift scenrio, the response ws reduced for three genertions with b 2 = 0.5, which is only one genertion more thn with the mximum b 2 = With b 2 = 0.5, the minimum responses were similr with GS nd GS + GE scenrios. Inbreeding Inbreeding ws clculted with the pedigree tht trced bck to the founders in genertion -5. In genertion 0, verge inbreeding F ws 0.9%. The increse in F ws lrger in genertions in which the selection emphsis on the desired llele ws strong. The biggest increse in F ws seen in the sme genertion or one genertion fter the lrgest decrese in selection response for the polygenic trit (Fig. 2). Genome editing reduced selection pressure on the desired llele nd therefore resulted in lower rtes of inbreeding in the GS + GE scenrio in the genertions in which the desired llele ws not fixed. Without selection on the desired llele (b 2 = 0), the inbreeding level ws 2.2% in genertion 5 (Tble 3) nd with moderte selection (b 2 = 0.5), the inbreeding level in genertion 5 ws only slightly higher t 2.3 or 2.4%. These inbreeding levels in genertion 5, with no selection or moderte selection on the desired llele, were very similr, regrdless of whether GE ws used or not (Tble 3). With moderte or no selection pressure on the desired llele, inbreeding levels were the sme fter 20 genertions of selection whether GS or GS + GE ws pplied. Only when ll the selection emphsis ws on the desired llele (b 2 = 1000), did GE decrese the long-term dditionl inbreeding from 1.2% with GS to 0.6% with GS + GE (Tble 4). Inbreeding ws lso ssessed in species-specific scenrios in which numbers of mles nd femles per genertion were chosen to be close to those of rel breeding progrms. Inbreeding levels without selection on the desired llele were 4.9, 12.2, 11.7, nd 31.4% in genertion 20 for the cttle, pig, fish, nd smll breeding scenrios, respectively. In scenrios with ll the selection emphsis on the desired llele, the long-term dditionl inbreeding ws 1.2, 2.1, 3.2, nd 4.9% with GS nd 0.6, 1.1, 1.4, nd 2.6% with GS + GE, respectively for the cttle, pig, fish nd smll breeding scenrios. The ddition of GE reduced the long-term dditionl inbreeding by pproximtely hlf in ll scenrios compred to GS lone. Costs nd benefits The cumultive benefit of incresing the frequency of the desired llele ws mesured s the percentge of nimls tht showed the desired phenotype, cross ll genertions fter genertion 0. For dominnt trit, this included nimls with the heterozygous nd with the desired homozygous genotype, nd for recessive trit, this only

6 Pge 6 of 14 GS GS + GE Desired llele frequency Delt G Delt F b Genertion Fig. 2 Response to selection. Frequency of the desired llele, chnge in G, nd F, in response to genomic selection (GS) nd in response to genomic selection with genome editing (GS + GE), pplying different weights (b 2 ) on the desired llele of monogenic trit included nimls with the desired homozygous genotype. Scenrios were compred t genertion 5 (Tble 3) nd genertion 20 (Tble 4). In sitution where fixtion of the desired llele is essentil in the short term, ll the weight cn be put on the desired llele. With GS, fixtion took four genertions (Tble 2) nd incurred loss in selection response for the polygenic trit of 31.3 months (Tble 4). In this cse, the number of nimls tht still showed the undesired phenotype before fixtion ws (100% 95.9%) 20 genertions 20,000 offspring = 16,400 nimls for dominnt llele (Tble 4). With GS + GE, the dditionl cost ws due to the GE of 3912 zygotes while the loss in selection response ws reduced by 59% from 31.3 to 12.9 months

7 Pge 7 of 14 Tble 3 Popultion prmeters fter five genertions of selection Method b 2 Reduced response Allele frequency c F d Number e Cumultive benefit f σ A Months b Dominnt (%) Recessive (%) GS , GS + GE Index weight for the genotype of the monogenic trit b Months of selection response lost for the polygenic trit in genertion 5, compred to the genetic level of genomic selection (GS) with b 2 = 0 c Frequency of the desired llele in genertion 5 d Men inbreeding coefficient in genertion 5 e Cumultive number of editing procedures over 5 genertions f Percentge of nimls, cumultive over the 5 genertions, with the desired phenotype when the desired llele is either dominnt or recessive Tble 4 Popultion prmeters fter 20 genertions of selection Method b 2 Reduced response Frequency c F d Number e Cumultive benefit f σ A Months b Dominnt (%) Recessive (%) GS , GS + GE Index weight for the genotype of the monogenic trit b Months of selection response lost for the polygenic trit in genertion 20, compred to the genetic level of genomic selection (GS) with b 2 = 0 c Frequency of the desired llele in genertion 20 d Men inbreeding coefficient in genertion 20 e Cumultive number of zygotes edited over 20 genertions f Percentge of nimls, cumultive over the 20 genertions, with the desired phenotype when the desired llele is either dominnt or recessive nd the number of nimls with undesired phenotypes ws equl to 9600 (41% less thn the 16,400 nimls with GS). With moderte weight (b 2 = 0.5), the desired llele ws fixed with GS fter 19 genertions (Tble 2), the long-term genetic loss fter 20 genertions ws 11.9 months (Tble 4), nd 58,800 nimls hd the undesired phenotype (dominnt desired llele). With GS + GE, the dditionl cost ws due to GE of 7072 zygotes while the loss in selection response ws reduced to 1.7 months (86% less thn 11.9 months with GS). The number of nimls with the undesired phenotype decresed to 15,600 (73% less thn the 58,800 with GS) for dominnt effect. The number of nimls with the undesired phenotype decresed by pproximtely fourfold with GS + GE compred to GS. Editing success rte nd editing survivl Both editing success rte (k) nd editing survivl (s) were ssumed to be equl to 1.00 for ll scenrios presented so fr. When k ws reduced from 1.0 to 0.10 (scenrio GS + GE with b 2 = 0.5), the required number of editing procedures lmost doubled (Tble 5), the selection response fell behind by 5 months (Tble 5), nd the time to fixtion doubled from four to eight genertions (Fig. 3). When s ws reduced from 1.0 to 0.05, the increse in number of editing procedures ws smller thn with reduced k, round 50% more insted of 100% (Tble 6), nd the time to fixtion lso incresed by 50% from four to six genertions (Fig. 4). When s ws low, surviving zygotes were lwys edited nd thus likely to be selected. When k ws low, non-edited offspring could be selected over their edited full-sibs, when their EBV for the polygenic trit ws sufficiently high.

8 Pge 8 of 14 Tble 5 Long-term impct of success rte of genome editing Success rte Months b Procedures c Cumultive benefit d Dominnt (%) , , , Recessive (%) Impct of editing success rte under moderte selection intensity for the desired llele combined with genome editing Success rte of editing n undesired llele into the desired llele b Long-term loss (mesured t genertion 20) in months of selection response for the polygenic trit, compred to success rte of 1.00 c Totl number of editing procedures d Percentge of nimls with the desired phenotype, cumultive over 20 genertions, when the desired llele is either dominnt or recessive While reduced k hd more impct on the number of zygotes tht needed to be edited, reduced s hd more impct on the loss in selection response. For k or s of 20%, the loss in selection response ws 5.3 months versus 7.4 months, respectively (Tbles 5 nd 6). Efficiency of genome editing To mimic the current low efficiency of GE [20], k of 0.20 ws combined with s of 0.20 in order to rech one successfully edited zygote for 25 edited zygotes. When these vlues were used in the GS + GE scenrio with b 2 = 0.5, fixtion of the desired llele ws obtined fter nine genertions with 12,144 zygotes edited (Tble 7). In comprison to the GS scenrio without GE, the number of nimls with the undesired phenotype before fixtion ws 26% less, nd the long-term loss in selection response ws 28% lrger. In comprison to complete editing efficiency, 72% more zygotes needed to be edited nd the loss in selection response ws 8 times higher (Tble 7). The impct of reduced efficiency ws reltively lrger on the loss of selection response thn on the incresed number of edited zygotes. With n efficiency of 4%, GE my enhnce the increse in frequency of the desired llele compred to genomic selection, but incresed the reduction in selection response due to reduced selection intensity. Discussion The objective of this study ws to investigte how GE in combintion with genomic selection could ccelerte the increse in frequency of the desired llele for monogenic trit compred to genomic selection lone. Assuming 100% ccurcy nd survivl llowed n ssessment of the potentil of the technology. We observed strong fvourble impct of GE on time to fixtion, loss in polygenic response, nd number of nimls tht hd the undesired phenotype before the desired llele ws fixed. The min results re summrized in Tble Desired llele frequency k Genertion Fig. 3 Response in frequency of the desired llele with reduced editing success. Allele frequency under genomic selection with genome editing (GS + GE) for different levels of editing success probbility (k)

9 Pge 9 of 14 Tble 6 Long-term impct of survivl rte of genome editing Editing survivl Months b Procedures c Cumultive benefit d Dominnt Recessive (%) (%) , Impct of editing survivl rte under moderte selection intensity for the desired llele combined with genome editing Survivl rte of zygotes subjected to editing b Long-term loss (mesured t genertion 20) in selection response for the polygenic trit, compred to complete survivl c Totl number of editing procedures d Percentge of nimls with the desired phenotype, cumultive over 20 genertions, when the desired llele is either dominnt or recessive Breeding progrms with genome editing In this study, the opportunities of pplying GE to increse the frequency of the desired llele of monogenic trit were evluted. Erlier results of simultions of breeding with GE showed tht polygenic trits could be improved by using PAGE [11]. An importnt hurdle to overcome for pplying PAGE to quntittive trits nd for incresing llele frequencies of monogenic trits is the vilbility of trget muttions to edit. Only smll number of cusl muttions re known tht ffect quntittive trits, nd lso the number of known muttions for monogenic trits is still limited. Mking the ppliction of GE costeffective with smll number of trgets tht hve known effects is more likely for monogenic trit thn for quntittive trit nucleotide (QTN) tht ffects polygenic trit. With PAGE, n incresed selection response of 4 to 8% ws reched by mking single edit in ech sire, provided tht ll the QTN were known nd different trgets could be used in different sires [11]. Alterntively, GE could be pplied to mny loci to simultneously prove nd use the effect of vrints [12]. With this pproch, the benefits from GE cn be much lrger thn those shown in simultion studies to dte, in which limited number of known QTN were ssumed vilble. When multiple edits cn be mde to the sme zygote t low cost, the integrted testing nd use of edits my mke GE on QTN of smll or uncertin effects cost-effective. Designs for these schemes need to be developed nd tested nd could give better results s unproven vrints my just be neutrl. Hving only one single trget my still be beneficil if its vlue for the popultion is lrge, s for instnce when single gene cn confer resistnce to disese. A monogenic trit, such s polledness, cn lso be highly relevnt in view of the costs nd the required chnges in niml welfre regultions for dehorning of cttle. A single geneediting trget tht is responsible for smll percentge of the genetic vrince of quntittive trit my hve limited vlue. The money needed to perform GE on such trget my result in bigger return when it is spend on 1.00 Desired llele frequency s Genertion Fig. 4 Response in frequency of the desired llele with reduced editing survivl. Allele frequency under genomic selection with genome editing (GS + GE) for different levels of editing survivl (s)

10 Pge 10 of 14 Tble 7 Popultion prmeters in genertions 5 nd 20 with n editing success of 0.20 nd survivl rte of 0.20 Genertion Reduced response Frequency c F d Procedures e Cumultive benefit f σ A Months b Dominnt (%) Recessive (%) , Index weight for the genotype of the monogenic trit ws b 2 = 0.5 b Months of selection response lost for polygenic BV compred to the genetic level of genomic selection (GS) with b 2 = 0 in the sme genertion c d Frequency of the desired llele Men inbreeding coefficient e Cumultive number of editing procedures up to the current genertion f Percentge of nimls with the desired phenotype, cumultive up to the current genertion, when the desired llele is either dominnt or recessive Tble 8 Summry of results on the impct of genome editing nd of reduced editing efficiency Efficiency (%) Mesure GS b GS + GE c Impct of GE d (%) Impct of efficiency e (%) 100 Time to fixtion (genertions) Loss in polygenic trit response (months) inbreeding level in genertion 20 (F) Undesired phenotypes (N) 58,800 15, Editing procedures (N) Polygenic trit response (months) Editing procedures (N) 12, Vlues from scenrios with moderte selection emphsis on the desired llele (b 2 = 0.5) Percentge of edited zygotes surviving to reproduction ge, 100% from k = 1, s = 1, 4% from k = 0.2, s = 0.2 (Tble 1) b Vlues from scenrio with genomic selection only c Vlues from scenrio combining genomic selection nd genome editing d Percentge chnge between columns GS nd GS + GE e Percentge chnge from GS + GE with 100% efficiency to GS + GE with 4% efficiency dditionl genotyping or phenotyping to increse selection response. Inbreeding In comprison to current breeding progrms, the observed rte of inbreeding in these simultions ws low, i.e. 0.25% per genertion, minly becuse of the lrge number of prents, in prticulr, the number of mles per genertion (100) ws lrger thn tht commonly used in livestock breeding progrms. The level of inbreeding incresed when emphsis of selection on the desired llele incresed (higher b 2 ), but not when GE ws dded to the scenrios. With b 2 = 1000, inbreeding rte ws ctully lower in GS + GE compred to GS scenrios (Tbles 3 nd 4). An increse in inbreeding rte my be observed when dding PAGE to GS in scenrios in which smll number of sires is edited [11]. In our simultions, edits were mde in zygotes from mny mtings, which requires more editing procedures, but in this wy, it ctully reduced inbreeding becuse the desired llele becomes vilble in mny more fmilies. To test the impct on inbreeding with smller popultion sizes, four species-specific scenrios were evluted. In ll those scenrios, the verge inbreeding rte fter 20 genertions ws similr in GS nd GS + GE scenrios, except with b 2 = Even with relistic number of prents per genertion, inbreeding rte ws still reltively low for diry cttle, pig nd fish breeding scenrios, rnging from 4.7 to 12.2% in genertion 20. This low inbreeding rte in our simultions is probbly becuse contributions of ll prents were ssumed equl. Therefore, scenrio with smll breeding progrm ws included tht resulted in n verge inbreeding level of 31.4% in genertion 20. Additionl inbreeding from selection on the desired llele incresed with smller effective popultion size but the benefit of using GS + GE insted of GS lso incresed, such tht the dditionl inbreeding ws hlved in ll scenrios by dding GE. Selection index The polygenic breeding vlue nd the frequency of the monogenic llele were selected for simultneously by combining the polygenic EBV nd monogenic genotype

11 Pge 11 of 14 in n index with fixed index weights. This index ws the sme for scenrios with nd without GE. In these simultions, the trde-off between chnging the llele frequency of the monogenic trit nd the genetic progress for the polygenic trit ws not optimized ginst genetic progress or inbreeding. Insted, three very different scenrios were included depending on the reltive emphsis tht ws given to the frequency of the desired llele. For the intermedite scenrios with b 2 = 0.5, optimiztion of the llele frequency trjectory could reduce the loss in genetic progress, nd lso the rte of inbreeding [28 31]. The weights for the polygenic trit nd the desired llele cn be optimized given the strting llele frequency nd the time t which fixtion is desired. Optimiztion of the chnge in frequency of the desired llele with selection cn reduce the disdvntge of GS scenrios in comprison to GS + GE. Alterntives for genome editing Introducing new lleles or incresing the frequencies of desired lleles does not necessrily require GE. Alterntives re introgression nd selection for the desired lleles. Disdvntges of introgression re potentilly lower genetic level of the donor breed nd lower genetic gin, linkge drg nd incresed inbreeding in the region surrounding the trget gene [13, 14]. In our simultions, we ssumed tht the desired llele ws lredy present in the popultion t strting frequency of The crriers of the desired llele were chosen t rndom nd therefore, on verge, t the sme genetic level s non-crriers. This ws n dvntge compred to introgression from nother (inferior) breed. This dvntge will lso pply when the desired llele needs to be introduced by GE. Moreover, by choosing the nimls with the highest EBV s trgets for GE, s ws done in the GS + GE scenrios, the desired llele cn ctully become fvourbly correlted with the polygenic trit. This is the opposite effect from linkge drg s observed with introgression nd selection, for which negtive correltion between the polygenic nd the monogenic trits my rise becuse of gmetic phse disequilibrium [26]. Genome editing offers cler benefits compred to lterntives when introducing new lleles or incresing llele frequencies from low vlues. Chllenges with genome editing Genome editing technology is being developed nd tested in nimls [6 8] but which procedures will be pplied in breeding progrms remins to be determined. Depending on the species, the use of reproductive technology is t different stges of implementtion. In diry cttle, in vitro production of embryos (IVP) nd multiple ovultion nd embryo trnsfer (MOET) re commonly used in breeding progrms, nd pplying GE during IVP my be reltively esy to implement. In this cse, the prents my be selected s cndidtes to produce edited offspring, s ws simulted in this study. An lterntive, s simulted by Jenko et l. [11], is to select the best nimls bsed on their breeding vlue nd pply GE to these elite nimls. Editing these elite nimls requires cloning procedure to produce n edited copy of the selected nimls such s somtic cell nucler trnsfer [20], unless the sme embryo cn be genotyped nd selected, nd subsequently edited before developing into n offspring. This procedure could tke plce in vitro by selecting the best embryos bsed on their genomic EBV nd subsequently using cells from the sme embryo to mke edited clones. The genertion intervl could increse if cloning step is needed, but this cn be kept to minimum if the whole procedure of genotyping, genomic selection, nd editing cn be done in vitro. In ll cses, in vitro reproduction is n essentil step to pply GE. Although in vitro reproduction techniques re frequently used in cttle, these techniques re much less developed in other mjor griculturl species such s pigs nd chickens. Moreover, if editing of individuls is desired fter obtining their EBV, insted of editing the offspring of selected prents, the use of cloning somewhere in the process my be necessry. Even when in vitro reproduction is n estblished technology, there re number of technicl issues to be solved with GE before lrge-scle ppliction is fesible. Currently, the probbility of obtining live edited niml from pplying GE is not high. Survivl of edited zygotes is one importnt fctor, but lso the technicl ccurcy of GE needs to be considered. Genome editing my result in mosics in which, prt of tissue is edited nd other prts re not. In ddition, GE my led to off-trget effects, i.e. chnges in the genome t other positions thn those intended. The occurrence of mosics nd off-trget effects cn be considered s n increse in mortlity, s simulted in this study, ssuming tht mosicism nd offtrget effects re either lethl or tht the nimls re not used for breeding for ethicl or sfety resons. However, while direct mortlity is cler outcome of procedure, excluding mosic embryos or embryos with off-trget edits requires their identifiction, which will be chllenging. Strtegies to screen for these effects re needed to void them going undetected [32 35]. A finl chllenge with GE is the uncertinty bout future cceptnce of ppliction of the technique in niml breeding. The legl frmework for its ppliction is not cler everywhere, which my limit the efforts put into reserch nd potentil pplictions. Some methods of ppliction require cloning of nimls, which is not llowed in severl countries. Clerly, n ethicl debte is

12 Pge 12 of 14 needed on when nd why the ppliction of GE in niml breeding is desirble. The potentil costs nd benefits presented in this pper cn contribute to this debte. Opportunities with genome editing When frequency of monogenic llele is incresed by GE lone, nd GE hs no detrimentl effects such s reduced survivl, breeding progrm would not suffer from lower selection response for the polygenic trit. For breeding progrms tht supply different mrkets (with potentilly different competitors), this llows complince with regultions tht pply for specific mrkets, for instnce where dehorning is not llowed, without reducing the competitiveness in other mrkets tht do not impose these restrictions. Determining the benefits of technology is not simple clcultion, since vlues cnnot lwys be cptured on the sme scle. Here, we mde n ttempt to compre such vlues for polledness in cttle using the results in Tble 3, for which the impct of different scenrios ws evluted fter five genertions of breeding. Over the course of five genertions, our simulted popultion counted 100,000 new-born nimls. The bsic scenrio ws GS with b 2 = 0, where no effort ws mde to reduce polledness. In this scenrio, 97.9% of clves, or 97,900 nimls, were born with horns nd would hve to be dehorned, which is costly nd pinful procedure for the clves. Assuming tht the cost of dehorning is 10 per niml, the totl cost of dehorning in our simulted popultion mounts to lmost one million fter five genertions. When pplying GE without selection in scenrio GS + GE with b 2 = 0, n dditionl 58.5% ( %) of the 100,000 new-born nimls would hve the polled phenotype, resulting in 585,000 sved fter five genertions. The ssocited cost ws the ppliction of GE in 10,000 zygotes. In this scenrio, there ws no negtive impct on selection response or on inbreeding, so the brekeven cost for pplying GE would hve been per zygote. In mny countries, the ntionl popultion of diry cttle is much lrger nd the 100 selected mles would be used to serve much lrger cow popultion, in which cse the brekeven cost of n edited zygote would become much higher. With cow popultion of 1 million nimls nd when evluting costs nd benefits fter five genertions, the brekeven cost for editing one zygote would be Editing trgets Genome editing hs the potentil to crete mjor chnge in how we perceive nd implement genetic improvement of livestock nd other species. While niml genetics reserch hs put lot of effort in the discovery nd investigtion of the effects of genomic vrition on phenotypes, its implementtion in breeding progrms hs been miniml. When GE becomes cost-effective nd ccepted technology, the use of individul vrints in breeding progrms my finlly become importnt. Such promises were lso mde for mrker-ssisted selection, but relity proved more difficult. The dvntges of GE re tht it does not rely only on vrints tht segregte in the trget popultion, nd, in cse of low llele frequency, it does not depend on the smll number of nimls in which the vrint is found. However, drwbck tht GE shres with mrkerssisted selection is tht in order to mke the best use of it, the trgets to edit must be known. Among the trgets described in the literture, re lleles of the RELA gene, which were shown to hve role in the resistnce to Africn swine fever in wrthog [36]. Introduction of these lleles by GE into domestic pigs ws recently reported [37]. Another exmple is deletion in the prolctin receptor gene tht determines the slick cot nd het tolernce trits in Senepol cttle [38]. Introduction of this llele into other cttle breeds by GE hs been suggested [38]. While there re some exmples of vrints tht cn be trgets for GE, such s those bove or the polled locus used here, the number of known trgets of interest to breeding schemes is currently limited. Thus, there is renewed need for identifying cusl muttions in the genome nd for predicting their effect on phenotypes. The number of GWAS studies in nimls is very lrge [39], but the resulting knowledge bout cusl muttions is so fr limited. While it remins difficult to determine cusl effects, vrints tht re present in rre breeds or loclly-dpted popultions will become more importnt. In the pst, these vrints were studied, but their introduction into commercil breeding popultions ws not considered due to the issues with introgression tht were discussed erlier. With GE, ll these vrints become of interest for breeding popultions, independent of their current genetic bckground. Conclusions In our simultion, we showed tht GE strongly decresed the time to fixtion for the desired llele compred to genomic selection lone. The loss in selection response in the polygenic trit ws much smller, up to sevenfold, with the ddition of GE. The sme level of inbreeding ws observed with or without GE, except when ll the selection emphsis ws plced on the monogenic trit; then GE reduced long-term inbreeding. Combining GE with moderte selection emphsis for the desired llele reduced by bout four-fold the number of nimls with the undesired phenotype over ll genertions. With relistic editing efficiency of 4%, the number of

13 Pge 13 of 14 required editing procedures incresed by 72% nd the loss in selection response incresed by 800%. With low efficiency, loss in selection response ws 29% more compred to genomic selection lone. In ddition to ethicl nd welfre considertions, creful ssessment of the technicl costs nd benefits of GE in commercil livestock is required. Additionl file Additionl file 1. Simultion_genome_editing.R. This file presents the simultion progrm in R to crete the simultion scenrios presented. Authors contributions HM conceived the study nd mnged the project. All uthors contributed to defining the reserch questions nd interprettion of the results. HM, HB nd JWMB defined the simultion models. JWMB progrmmed nd crried out the simultions nd performed the dt nlyses. JWMB nd HM drfted the mnuscript. All uthors red nd pproved the finl mnuscript. Competing interests The uthors declre tht they hve no competing interests. Avilbility of dt nd mterils Only simulted dt ws used in this study. Simulted dtsets cn be reproduced with the simultion code provided in Additionl file 1. Ethics pprovl nd consent to prticipte Not pplicble. Funding This study ws finncilly supported by the Dutch Ministry of Economic Affirs (TKI Agri & Food) nd the Breed4Food prtners Cobb-Europe, CRV, Hendrix Genetics nd TopigsNorsvin. Publisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in published mps nd institutionl ffilitions. Received: 29 My 2017 Accepted: 2 April 2018 References 1. Ledford H. Slmon pprovl herlds rethink of trnsgenic nimls. Nture. 2015;527: Ledford H. Trnsgenic slmon ners pprovl. Nture. 2013;497: Yin H, Xue W, Chen S, Bogord RL, Benedetti E, Grompe M, et l. Genome editing with Cs9 in dult mice corrects disese muttion nd phenotype. Nt Biotechnol. 2014;32: Oskbe Y, Oskbe K. Genome editing with engineered nucleses in plnts. Plnt Cell Physiol. 2015;56: Tn W, Crlson DF, Lncto CA, Grbe JR, Webster DA, Hckett PB, et l. 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