
 4cl4-coumarate : coa ligasecas9crispr - associated protein 9crisprclustered regularly interspaced short palindromic repeatsctcondensed tanningrnaguide rnammillionpamprotospacer adjacent motifpol iiirna polymerase iiirnairna interferencernaseribonucleasesnpsingle nucleotide polymorphisms / g ligninsyringyl - to - guaiacyl lignin ratiotrnatransfer rna 4-coumarate : coa ligase crispr - associated protein 9 clustered regularly interspaced short palindromic repeats protospacer adjacent motif single nucleotide polymorphism syringyl - to - guaiacyl lignin ratio
since the first report of its programmable gene editing potential , the crispr / cas9 technology is revolutionizing all facets of biology from medicine to agriculture ( jinek et  al . , 2012 ) .
with its efficiency and simplicity , crispr / cas9 has quickly displaced its predecessors ( e.g. , zinc finger and transcription activator - like effector nucleases ) as the method of choice for genome editing ( carroll , 2014 ) . for agricultural applications that have traditionally depended on gene - silencing for rna modification , crispr / cas9 is a game changer .
the previous methods , such as antisense or rna interference ( rnai ) , leave much to be desired as the degree , specificity and stability of gene silencing are not always predictable .
this necessitates screening and characterization of a large number of transgenic lines for the desired trait .
by contrast , crispr / cas9 is guided to disrupt the reading frame  thereby protein function  of the target gene by dna editing .
2012 ) , and both monoallelic and biallelic mutations are reported ( bortesi and fischer , 2015 ) . with biallelic editing , null mutations are readily obtained in primary transformants , though with varying efficiencies ( brooks et  al . , 2014 ; zhang et  al . , 2014 ; zhou et  al . , 2014 , 2015 ) ,
interestingly , the most efficient and consistent crispr / cas9 editing was reported in hybrid populus tremula x alba clone 717 - 1b4 ( 717 ) , with biallelic mutations detected in all independent transgenic lines examined ( zhou et  al . , 2015 ) .
for woody perennials such as forest trees , fruit / nut trees and woody ornamentals with highly heterologous genomes and long generation times , crispr / cas9 affords a facile means to accelerate genetic improvement .
the first application of crispr / cas9 genome editing in populus targeted the 4-coumarate : coa ligase ( 4cl ) gene family involved in phenylpropanoid metabolism ( zhou et  al . , 2015 ) .
one of the genes , 4cl1 ( potri.001g036900 ) , has been extensively characterized for its involvement in lignin biosynthesis .
down - regulation of 4cl1 or its orthologs leads to reduced lignin content and altered lignin structure in a number of species ( boerjan et  al . ,
thus , crispr / cas9 editing of 4cl1 served as a proof - of - concept study , allowing an assessment of its efficacy in perturbing lignin biosynthesis relative to previous methods .
we generated 36 independent transgenic lines , and amplicon - sequencing of randomly selected events confirmed biallelic mutations in all cases ( zhou et  al . , 2015 ) .
as frequently reported for crispr / cas9 genome editing , small indels , especially 1-bp modifications , were the predominant patterns and predicted to disrupt the reading frame .
consistent with this prediction , lignin content was reduced by 23% with a concomitant decrease of s / g lignin ratio by 30% in all transgenic plants ( zhou et  al . , 2015 ) .
wood discoloration is a known side effect of lignin modification frequently observed in transgenic plants with suppressed lignin gene expression .
however , the coloration patterns reported in those cases lack the uniformity we observed for the crispr / cas9 mutants . for example
, antisense downregulation of 4cl1 in populus resulted in patchy wood discoloration in only 5 of 14 transgenic lines ( voelker et  al . ,
wood discoloration patterns of transgenic populus with down - regulated expression of cinnamoyl - coa reductase were highly variable among vegetatively propagated plants ( van acker et  al . , 2014 ) , presumably due to the unstable nature of sense- or antisense - mediated post - transcriptional gene silencing .
rnai - mediated gene suppression is considered a more effective alternative , with improved trait stability ( li et  al . , 2008 ) .
for instance , rnai - silencing of 4-coumaroyl - coa 3-hydroxylase in transgenic populus resulted in lignin reductions among 9 independent events that ranged from no change to 1560% ( coleman et  al . ,
similarly , rnai silencing of 4cl in pinus led to wood discoloration in only 2 of 12 viable transgenic lines ( wagner et  al . , 2009 ) .
by contrast , the homogeneity of wood discoloration both within and across independent crispr / cas9-edited populus mutant lines is consistent with a null 4cl1 , and resembles that of naturally occurring  brown midrib  mutants of maize and sorghum ( sattler et  al . , 2010 ) .
the results serve as testament to the superiority of crispr / cas9 over previous gene silencing methods . 

( 2015 ) with permission . a second gene , 4cl2 ( potri.019g049500 ) that is phylogenetically distinct from lignin - associated 4cls ( chen et  al . , 2014 ) .
has long been suggested to participate in flavonoid biosynthesis , based solely on its preferential expression in epidermis and roots ( harding et  al . , 2002 ) .
crispr / cas9 mutation of 4cl2 was therefore expected to yield functional evidence to substantiate this role . as with the 4cl1 case
, biallelic mutations were observed in all independent transgenic lines surveyed by amplicon - sequencing ( zhou et  al . ,
the metabolic consequence was examined in roots where flavonoid - derived condensed tannins ( cts ) are known to accumulate to high levels ( 20% dry weight ) in the experimental poplar genotype ( chen et  al . , 2014 ) .
crispr / cas9 mutations of 4cl2 resulted in significantly reduced levels of condensed tannins in roots , providing reverse genetics evidence in support of class ii 4cl function in flavonoid biosynthesis .
after our initial publication , successful crispr / cas9 editing was reported for populus tomentosa ( fan et  al . , 2015 ) .
that study targeted a phytoene desaturase ( pds ) gene for mutagenesis by multiplexing 4 grnas , and observed a 50% frequency of albino phenotypes .
the lower mutation rates compared to our study may be attributed to gene redundancy and sequence heterozygosity that were not accounted for during grna design ( see discussion below ) .
regardless , selected albino transgenic lines were sequence - confirmed to harbor biallelic mutations ( fan et  al . , 2015 ) , providing additional support for the superior phenotypic uniformity of crispr / cas9 mutants .
a major concern in genome editing is the specificity of the dna modification , as off - target cleavage due to non - specific crispr / cas9 activity may cause unintended mutations .
this is especially true for plant genomes that are characterized by recurring episodes of whole - genome , segmental and tandem duplications .
we assessed crispr / cas9 specificity in populus by analyzing its on - target versus off - target activities among duplicated genes .
the grna for 4cl1 was designed to discriminate against the paralogous 4cl5 ( potri.003g188500 ) , with 3 mismatches in the target sequence and one mismatch in the protospacer adjacent motif ( pam ) ( zhou et  al . , 2015 ) .
by designing consensus primers flanking the target site , we were able to sequence both 4cl1 and 4cl5 amplicons simultaneously to assess the specificity of crispr / cas9 .
a custom program , ageseq ( analysis of genome editing by sequencing ) , was developed to facilitate variant detection ( xue and tsai , 2015 ) .
no off - target cleavage in the 4cl5 locus was detected in 4cl1-edited lines , suggesting that crispr / cas9 editing in populus is highly specific ( zhou et  al . , 2015 ) .
negative  experiment , where mismatches between a 4cl5-grna , designed based on the p. trichocarpa reference genome ( phytozome v3 ) , and the corresponding sequence of p. tremula x alba 717 used for transformation abolished cleavage in all transgenic lines examined ( zhou et  al . , 2015 ) .
in this case , the mismatches were in the form of 2 single nucleotide polymorphisms ( snps ) , one per allele , located near or in the pam .
these results , together with the highly heterologous nature of woody perennials , suggest that off - target cleavage of crispr / cas9 is likely to be low in these species .
the ability of crispr / cas9 to discriminate single - base differences makes it a powerful tool to investigate functional redundancy of highly homologous genes that are otherwise difficult to discern using gene silencing approaches .
for instance , 4cl1 is the most abundantly expressed isoform in populus xylem , constituting > 85% of 4cl transcripts , with 4cl5 being a distant second , based on rna - seq data ( swamy et  al . , 2015 ) .
however , the crispr / cas9 knockout of 4cl1 reduced lignin content only by 23% ( zhou et  al . , 2015 ) . because the 4cl5 locus was unmodified ,
it is likely involved in the residual lignin biosynthesis of the 4cl1-knockout mutants . in the populus study that targeted pds ,
4 grnas were designed based on one gene model ( potri.014g148700 ) without considering the genome duplicate ( potri.002g235200 ) that shares a high degree ( 93% ) of coding sequence identity . while the genome sequence of p. tomentosa is not yet available , analysis of the p. trichocarpa and p. tremula x alba orthologs revealed different degrees of sequence variation between the 2 duplicates in the grna target regions .
these differences ( plus unknown allelic heterozygosity ) likely contributed to the variation in the reported editing efficiencies of different grnas ( fan et  al . , 2015 ) .
examples include designing multigene - targeting grnas based on conserved sequences of homologous genes ( brooks et  al . , 2014 ; jacobs et  al . , 2015 ) , or multiplexing individual grna cassettes ( brooks et  al . , 2014 ; fan et  al . , 2015 ; zhang et  al . , 2014
a recent study hijacked the trna processing machinery for efficient processing of up to 8 , and potentially more , grnas from one single construct ( xie et  al .
, the grna is positioned downstream of trna as a polycistronic gene with repeating trna - grna units for multiplexing ( xie et  al . , 2015 ) . because mature grnas are released following cleavage of the polycistronic transcript by the trna processing machinery
, there is no sequence constraint at the 5 end of grnas ( xie et  al . , 2015 ) .
this greatly simplifies grna design , as any sequences upstream of pam ( ngg ) can be used ( e.g. , n20-ngg or other truncated lengths ( fu et  al . , 2014 ) ) , unlike with the standard design where grna is transcribed directly from the pol iii u6 or u3 promoter using a specific transcription initiation sequence ( e.g. , gn19-ngg or an19-ngg , respectively ) ( wang et  al . , 2008 ) .
in some cases , grna stacking , either by re - transformation or via controlled crosses of primary transformants may be necessary in order to discern individual gene functions .
as discussed above , numerous studies have exploited sequence polymorphisms for developing gene- or allele - specific grnas to achieve precision editing by crispr / cas9 .
however , few studies have addressed the added complexity of sequence polymorphisms on crispr / cas9 genome editing , probably because the common plant models , such as arabidopsis , rice , soybean and tomato , are selfing species with homozygous genomes .
we argue that sequence polymorphisms are of concern in genome editing of most flowering plants , especially woody perennials that are outcrossing in nature .
this is supported by the high frequency of genic snps reported for trees , one per 60  bp in populus tremula ( ingvarsson , 2005 ) or one per 16 - 33  bp in eucalyptus depending on the species ( klheim et  al . , 2009 ) .
a recent population genomics study of 544 populus trichocarpa individuals identified 17.9 million ( m ) snps by whole - genome resequencing ( evans et  al . , 2014 ) . using a similar approach with both resequencing and rna - seq data
, we uncovered over 10  m snps from a single individual , p. tremula x alba clone 717 that is commonly used in populus transformation ( xue et  al . , 2015 ) .
given the high levels of heterozygosity in tree species , the need to consider sequence polymorphisms in genome editing experiments can not be over - emphasized .
in fact , most published genome sequences are , by default , consensus sequences of their ( pseudo)haploid genomes .
thus , while several web - based grna design programs are available for plants , such as crispr - plant ( xie et  al . , 2014 ) . and
crispr - p ( lei et  al . , 2014 ) , their utility for outcrossing species is limited because the preloaded genome sequences lack biallelic ( or multiploid ) coverage . furthermore , it is often the case as in populus that the species or genotype chosen for whole genome sequencing ( e.g. , , p. trichocarpa nisqually-1 ) is different from that routinely used for genetic transformation ( e.g. , p. tremula x alba 717 ) . to facilitate crispr / cas9 genome editing in 717
, we constructed a custom 717 genome by substituting > 10  m sequence variants into the p. trichocarpa reference genome ( xue et  al . , 2015 ) .
we then assessed the impact of sequence polymorphisms on grna specificity for 1000 randomly selected genes .
grnas were designed based on the haploid genome of ( 1 ) p. trichocarpa or ( 2 ) 717 , and then cross - checked for snps or indels in the 717 target sequences . from the first analysis , we found that more than 57% of the grnas designed based on the p. trichocarpa genome correspond to regions that harbor snps or indels in 717 ( xue et  al .
the rates were slightly lower but nevertheless high ( 42 - 44% ) in the second analysis when the custom 717 genome was used , owing to allelic variations ( zhou et  al . , 2015 ) .
together , these data suggest that standard grna designs may suffer from a high probability of failure in crispr / cas9 genome editing , due to the frequent occurrence of snps / indels in outcrossers .
the 717 sequences and associated blast and gene model query tools are available via aspendb ( http://aspendb.uga.edu/s717 ) .
tool is particularly useful for screening custom grnas or pcr primers against known snps / indels in the p. trichocarpa or the custom 717 genome ( xue et  al . , 2015 ) .
a set of pre - selected grnas with no known sequence variants for 38,509 populus genes is provided ( zhou et  al . , 2015 ) .
the modified , variant - sensitive pipeline for grna design , called  aspen crispr designer ,  is also available at aspendb and can be applied to other species . for outcrossing species lacking a genomic variant database , several approaches can be applied to identify snps / indels in the gene(s ) of interest prior to grna selection .
rna - seq data from the transformation genotype , when available , is an excellent source for high - confidence snp discovery .
provided that the gene of interest is reasonably expressed ( e.g. , , with a high read coverage ) , manual inspection of candidate grnas against rna - seq alignments using programs such as the popular integrative genomics viewer ( http://www.broadinstitute.org/igv ) is usually effective to confirm their specificity . in the absence of deep - sequencing data , pcr primers ( with or without degeneracy
) can be designed to amplify the target gene coding region based on a closely - related reference genome , or on conserved amino acid sequences of orthologs as in conventional rt - pcr cloning ( hu et  al . , 1998 ) .
as pam ( ngg)the only sequence constraint in grna design  occurs frequently , it is not necessary to obtain full - length gene sequences .
however , the use of multiple primer pairs is recommended to increase the likelihood of capturing both alleles .
direct sequencing of pcr products to identify sequence variants is an established practice in molecular ecology research , and several programs have been developed to decode overlapping sanger chromatograms ( dmitriev and rakitov , 2008 ) . for researchers with convenient access to high - throughput sequencing ,
sequence reads can then be processed by ageseq to search for potential variants in the target gene sequences ( xue and tsai , 2015 ) .
ageseq is available as a standalone program or a galaxy - compatible tool ( https://toolshed.g2.bx.psu.edu ) to support web - based data analysis .
it should be noted that pcr amplification of noncoding sequences can be tricky due to the lower degree of sequence conservation ( xue et  al . , 2015 ) .
thus , for crispr / cas9 applications that target promoter or other noncoding sequences ( e.g. , transcriptional activation or chromatin modification ( sander and joung , 2014 ) ) , resequencing of the transformation clone for unbiased variant calling , as discussed above for populus 717 ( xue et  al . , 2015 ) , is recommended .
the populus study extended the ever growing list of plant species successfully genome - edited by crispr / cas9 ( bortesi and fischer , 2015 ; kumar and jain , 2015 ) to woody perennials .
woody perennials suffer as experimental systems due to the lengthiness of genetic transformation and plant characterization .
many forest and horticultural tree species and genotypes are notoriously recalcitrant to tissue culture regeneration with low transformation efficiencies ( litz and padilla , 2012 ; busov et  al . , 2005 ) .
limitations associated with other established technologies , such as incomplete silencing , position effects or epigenetic modifications of transgenes ( matzke and matzke , 1998 ) , further impede progress in transgenic tree research . even for species with a facile transformation system , such as populus tremula x alba 717 , the common practice of generating multiple independent events for preliminary screening prior to in - depth characterization of selected lines is resource - demanding and time - consuming ( fig .  2 ) .
the ability of crispr / cas9 to generate null mutations in primary transformants , and the promise of phenotypic consistency among independent biallelic mutants as demonstrated in the populus study make a compelling argument that it may soon become acceptable to characterize only a few transgenic lines with sequence - verified mutations .
high - throughput methods for mapping genome editing patterns ( xue and tsai , 2015 ) and transgene ( e.g. , t - dna harboring cas9 , grna and selectable marker ) insertion sites ( kanizay et  al . , 2015 )
are expected to facilitate this effort . as depicted in figure  2 , primary transformants with confirmed editing can be used directly for in - depth characterization when multiple independent events are available as biological replicates ( zhou et  al . , 2015 ) .
even when only a few mutation events are obtained ( for difficult - to - transform species or from suboptimal transformation trials ) , the ability to bypass rna - level screening and to obtain stable mutations still confers advantages over the gene - silencing approaches .
crispr / cas9 thus offers the enticing prospect of streamlined transgenic characterization and expedited large - scale functional characterization . 

figure 2.schematic comparisons of experimental approaches involving gene silencing vs. crispr / cas9 genome editing .
( a ) for gene silencing by antisense , sense or rnai approaches , knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation .
expression knowledge is not necessary for crispr / cas9 editing at the dna level , and vectors with a pol iii promoter ( e.g. , u6 or u3 ) for grna expression and a cas9 under control of a constitutive ( e.g. , 35s ) promoter are widely applicable .
( b ) following regeneration of putative transgenic plants , conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired ( or maximum ) levels of gene suppression .
it is common to screen a large number of events ( 10 ) to identify a minimum of 3 for subsequent analysis .
this process can be greatly simplified with crispr / cas9 mutants , as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis .
( c ) for species with a robust transformation system , multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . in case of difficult - to - transform species for which few biallelic events may be obtained ,
micropropagation is necessary to obtain biological replicates before in - depth characterization , as is typically done using gene silencing . 
 
( a ) for gene silencing by antisense , sense or rnai approaches , knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation .
expression knowledge is not necessary for crispr / cas9 editing at the dna level , and vectors with a pol iii promoter ( e.g. , u6 or u3 ) for grna expression and a cas9 under control of a constitutive ( e.g. , 35s ) promoter are widely applicable .
( b ) following regeneration of putative transgenic plants , conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired ( or maximum ) levels of gene suppression .
it is common to screen a large number of events ( 10 ) to identify a minimum of 3 for subsequent analysis .
this process can be greatly simplified with crispr / cas9 mutants , as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis .
( c ) for species with a robust transformation system , multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . in case of difficult - to - transform species for which few biallelic events may be obtained ,
micropropagation is necessary to obtain biological replicates before in - depth characterization , as is typically done using gene silencing .
biallelic mutations of the target gene(s ) in conjunction with the hemizygous nature of transgene ( e.g. , t - dna ) integration in primary transformants ( t0 ) means that dna - edited but transgene - free progenies can be readily obtained from t1 segregants , as demonstrated in annual species ( zhang et  al .
trait stacking with early flowering induction is thus highly desirable , for example by over - expression of flowering locus t ( ft ) as demonstrated in prunus ( plum ) , populus and eucalyptus ( hoenicka et  al . , 2014 ;
another concern for outcrossing species is that the progenies will be different from the parents , which necessitates additional screening and selection .
we note , however , that introgressing transgenic / mutant events into elite genotypes is an established practice in crop and tree breeding programs , and hence not unique to crispr / cas9-edited mutants .
a well - known tree example is the loblolly pine ( pinus taeda ) clone 756 that harbors a naturally occurring null allele of cinnamoyl alcohol dehydrogenase involved in lignin biosynthesis , and that is extensively used as a parent in several breeding programs in the us ( gill et  al . , 2003 ) .
another example is early - flowering ft - plum that serves as a parent in the fasttrack breeding program to expedite development of improved commercial cultivars ( callahan et  al . ,
various molecular and genomics tools are available to facilitate progeny selection in transgenic breeding programs .
recently , virus - based systems have been explored for transient expression of genome - engineering reagents ( ali et  al .
however , such systems still utilize t - dna to express the repurposed viral genome components , and the resulting plants are not transgene - free without crossing .
these methods also depend on agroinfiltration to deliver the vector into plant cells , which unfortunately is not applicable to most woody species with an open minor vein structure in their leaves ( gamalei , 1989 ) . clearly ,
development of innovative methods will be necessary for facile removal of t - dna from the genome - edited plants without crossing .
regardless , null segregants of crop cultivars derived from genome editing are indistinguishable from naturally occurring or chemically - induced mutants , and may therefore be exempt from gmo regulation in countries that adhere to product - based regulations ( voytas and gao , 2014 ) .
this is expected to significantly reduce the timeline and financial burden associated with developing new crop varieties , thereby encouraging adoption of crispr / cas9-based genome editing for the forest products , fruit / tree nuts , and woody ornamental industries .
the research in the tsai laboratory discussed in this article was supported by the department of energy , office of biological and environmental research ( grant no .
de - sc0008470 ) , the department of agriculture , national institute of food and agriculture ( grant no .
2015 - 67013 - 22812 ) , and by the georgia research alliance - hank haynes forest biotechnology endowment .
 abstractthe crispr / cas9 technology is a welcome breakthrough for genome editing , owing to its precision , efficiency , versatility and ease of adoption . 
 we recently reported the first application of crispr / cas9 for biallelic mutations in stably transformed populus , extending the species range of this powerful technology to woody perennials . 
 an underappreciated obstacle in genome editing of outcrossing species is the frequent occurrence of sequence polymorphisms that can render crispr / cas9 unproductive . 
 we discuss experimental evidence as well as genome - wide computational analysis to demonstrate the sensitivity of crispr / cas9 to allelic heterozygosity , and highlight tools and strategies that can help deal with such sequence polymorphisms . with its specificity , crispr / cas9 offers a less equivocal means than previous approaches for discerning functional redundancy of paralogous genes that are prevalent in plant genomes . 
 continuing improvements of the crispr / cas9 system for multiplex genome engineering should facilitate these efforts . 
 the paradigm shift brought about by crispr / cas9 promises to accelerate not only basic research but also applied crop improvement progress . 
ABBREVIATIONS
None
HIGH-EFFICIENCY GENOME EDITING OF 
PRECISION EDITING BY CRISPR/CAS9
HARNESSING SEQUENCE POLYMORPHISMS OF OUTCROSSING SPECIES FOR EFFICIENT CRISPR/CAS9 EDITING
CONCLUDING REMARKS AND FUTURE PROSPECTS
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Funding