with the advent of genome sequencing , there has been an explosion in the number of organisms being used for research into the evolution of gene regulation and function ( e.g. , emlen et al . 2005 ) or that might be adapted for these studies in the near future ( e.g. , kiontke et al . however , the only method available for analyzing gene function in many species is rna interference . despite the simplicity of this approach , there can be significant variations in the response to rnai between species ( nuez and felix 2012 ) , which complicates evolutionary comparisons . the analysis of null mutations would provide a better method for comparing the functions of genes across species , but creating these mutations can be laborious , and maintaining them often requires balancers . in addition , forward genetic screens would allow the unbiased identification of new genes or genes with unexpected functions , which can be critical for evolutionary analysis ( guo et al . 2009 ) . however , the ability to carry out these screens depends on genetic markers , balancing chromosomes and tools that took decades to develop for drosophila melanogaster or caenorhabditis elegans . for example , the related nematode c. briggsae has been studied for years , but random screens have only produced convenient sets of markers for half of its chromosomes . we show that transcription activator - like effector nuclease ( talen ) technology can overcome this problem . we used talens to help develop the model organism c. briggsae for genetic and evolutionary studies . talens can be designed to target any dna sequence ( reviewed by sun and zhao 2013 ) , and were recently tested in both c. elegans and c. briggsae ( wood et al . however , each talen requires 1520 repeat units to bind a unique target , so we adapted golden gate shuffling to streamline the process of making custom talens for nematodes . furthermore , new mutations can be difficult to study unless other genetic resources are available , so we used information from a set of c. elegans null mutations to identify and target genes needed to create markers , balancers , and tools for c. briggsae . to simplify producing custom talens for c. briggsae , we adapted the golden gate shuffling method ( cermak et al . s1 , supplementary material online ) . furthermore , to speed up the identification of new mutations , we screened for insertions or deletions by their altered size on 10% acylamide gels , eliminating the need for nuclease digestion ( fig . . following injection of talens targeting the cbr - him-8 gene , we isolated an f1 animal with the him phenotype ( high incidence of male progeny ) . dna from eight f2 self - progeny was amplified by the polymerase chain reaction ( pcr ) and separated on a 10% acrylamide gel , revealing the presence of two different alleles . dna from potential cbr - unc-51 mutants was amplified using four sets of primer pairs ( a  d ) . mutant cbr - unc-51(v215 ) lacked dna in the region of primer pairs c and d. ( c ) the primer pair e could amplify a single band of approximately 3 kb from v215 dna , indicating that most of this region was deleted . ( d ) map of the region , showing the extent of the 9423 bp v215 deletion , determined by sequencing the fragment shown in ( c ) . ( a ) many f1 animals were heterozygous for two distinct mutations . following injection of talens targeting the cbr - him-8 gene , we isolated an f1 animal with the him phenotype ( high incidence of male progeny ) . dna from eight f2 self - progeny was amplified by the polymerase chain reaction ( pcr ) and separated on a 10% acrylamide gel , revealing the presence of two different alleles . dna from potential cbr - unc-51 mutants was amplified using four sets of primer pairs ( a  d ) . mutant cbr - unc-51(v215 ) lacked dna in the region of primer pairs c and d. ( c ) the primer pair e could amplify a single band of approximately 3 kb from v215 dna , indicating that most of this region was deleted . ( d ) map of the region , showing the extent of the 9423 bp v215 deletion , determined by sequencing the fragment shown in ( c ) . positions on the map are related to the start of cbr - unc-51 . using these approaches , we created hundreds of mutations in dozens of genes during the past 8 months ( table 1 , unpublished results ) . the efficiency of some talens was so high that many f1 animals were heterozygous for two new mutations : one induced in the maternal genome and a second in the paternal genome after fertilization ( fig . although most of the mutations were small insertions or deletions , we occasionally recovered large deletions ( fig . thus , talens can produce a variety of mutations in nematode genes , and the procedure is rapid and efficient . 
 table 1.comparison of caenorhabditis elegans and c. briggsae mutations.genelgproteincel allelecel phenotypecbr allelecbr phenotype # cbr allelesunc-40inetrin receptor ( 1,415 aa ; chan et al . 1996)e1430 r157stopweak kinker unc , slightly dpyv248fs ( 8 bp )kinker unc , slightly dpy2dpy-5iprocollagen ( 284 aa ; thacker et al . 2006)e61 g203stopstrong dpyv234fs ( 8 bp /6 bp ins)strong dpy28smg-5inovel ( 549 aa ; anders et al . 2003)r860 q17stopnmd defective , pvulv246fs ( 8 bp )nmd defective , pvul4him-8izinc fingers ( 361 ; macleod et al . 1981)tm611 deletionhigh incidence of malesv188fs ( 7 bp )high incidence of males9unc-54imyosin heavy chain ( 1,963 aa ; macleod et al . 1981)e1092paralyzedv139fs ( 11 bp )paralyzed , lethal2q1072stopuncv138 ( 6 bp )paralayzed uncunc-34venabled / vasp ( 468 aa ; yu et al . 2002)gm104 w10stopcoiler uncv255fs ( 11 bp )coiler unc7dpy-11vthioredoxin - like ( 246 aa ; ko and chow 2002)e207 r4stopdpyv241 ( 6 bp )dpy11unc-51vprotein kinase ( 856 aa ; ogura et al . 1993)e5 q96stopkinker uncv272fs ( 11 bp )kinker unc3note.if a gene produces more than one transcript , only the size of the largest product is listed . a representative null alleles or strong loss - of - function allele is shown for each gene . cel alleleputative null alleles of each c. elegans gene except dpy-8 , which lacked data . note.if a gene produces more than one transcript , only the size of the largest product is listed . a representative null alleles or strong loss - of - function allele is shown for each gene . cel alleleputative null alleles of each c. elegans gene except dpy-8 , which lacked data . , we asked whether talens could be used to speed up the development of genetic model organisms , using c. briggsae as a test case . the first mutations in this species were isolated more than 60 years ago ( nigon and dougherty 1950 ) , but three of the six chromosomes still lacked sets of mapped marker genes to simplify genetic analysis . these three chromosomes ( lg i , v , and x , 
 www.briggsae.org , last accessed november 19 , 2013 ) contain about 50 mbp of dna . fortunately , bioinformatic resources for c. briggsae are extensive . 2003 ) , and single nucleotide polymorphisms between the af16 and hk104 strains have been used to create a rough linkage map ( hillier et al . these steps are critical for work in new species , but further progress depends on genetic resources . unfortunately , forward screens have produced few visible markers , and new mutations take time to place on the genetic map . reverse genetic approaches might solve this problem , but directed screens for large deletions have been slow and expensive ( hill et al . thus , we tested two criteria for selecting potential marker genes in c. briggsae : 1 ) they must be orthologs of c. elegans genes with viable null mutations and 2 ) the c. elegans mutants must have visible phenotypes . for example , c. elegans unc-32(e189 ) is a widely used marker for lgiii , but unc-32 null alleles are lethal ( pujol et al . similarly , we avoided genes with wild - type null phenotypes , such as unc-93 ( greenwald and horvitz 1980 ) . after selecting potential markers , we targeted each gene with talens , to see whether these guidelines allowed an accurate prediction of phenotypes in c. briggsae ( table 1 ) . this effort represents one of the largest cross - species comparisons of animal gene function done using targeted knockouts . using this approach the phenotypes of most mutants strongly resemble those of their c. elegans orthologs , including unc-40 and dpy-5 on lgi , unc-34 and unc-51 on lgv and unc-1 , dpy-8 , and unc-7 on x ( table 1 ) . however , the phenotypes of two genes were more severe  c . briggsae unc-54(v139fs ) and dpy-11 null mutants were barely viable and could not be maintained , whereas the corresponding c. elegans mutants were much healthier . in these two cases , we also recovered partial loss - of - function mutations that were viable and easily scored . overall , our approach predicted the null phenotypes for seven of nine genes and produced useful mutations for all nine . moreover , each new mutation was already positioned in the c. briggsae genome , providing an instant link between the physical and genetic maps ( fig . the approximate positions in centimorgans of snps mapped by bulk segregant analysis are shown above each line in parentheses ( koboldt et al . the locations of mutations in genes that affect vulval development , fertility , or dauer formation are shown in gray ( inoue et al . 2007 ; seetharaman et al . 2010 ; beadell et al . 2011 ; sharanya et al . 2012 ) . the approximate positions in centimorgans of snps mapped by bulk segregant analysis are shown above each line in parentheses ( koboldt et al . the locations of mutations in genes that affect vulval development , fertility , or dauer formation are shown in gray ( inoue et al . 11 ( kiontke et al . 2011 ) , another hermaphroditic nematode . after identifying the c. sp . s2 , supplementary material online ) , we used talens to induce v277 and v279 , two frameshift mutations that are null alleles . the mutant animals have difficulty moving normally , often coil , and show progressively greater defects with age , because the head bends to one side . these phenotypes are very similar to those of c. elegans unc-23 mutants , which cause fragile muscle attachments ( plenefisch et al . although mutants with visible phenotypes can be used as balancers or to mark chromosomes in crosses , other genetic tools are also important . first , many talen mutants produce truncated proteins because of frameshifting mutations . if these transcripts are not eliminated by the nonsense - mediated decay ( nmd ) surveillance system ( weischenfeldt et al . thus , we used talens to knock out c. briggsae smg-5 , which encodes a component of the nmd system ( anders et al . as predicted , this mutation restores partial activity to stop mutants in other genes ( manuscript in preparation ) . second , hermaphroditic populations of nematodes reproduce by self - fertilization , but males are essential for crosses or phenotypic analysis . thus , we created mutations in c. briggsae him-8 , which controls x - chromosome pairing ( phillips and dernburg 2006 ) . following self - fertilization , 35% of the cbr - him-8 progeny were xo males and only 2% died as embryos ( n = 498 ) . these phenotypes are similar to those for c. elegans him-8 ( hodgkin et al . 1979 ) . taken together , our results demonstrate a new use for talen or crispr ( damian and porteus 2013 ) technologies  developing new species as genetic model organisms . this approach should be particularly valuable for species that are easily cultured in the laboratory and have closely related model organisms to guide the work , like members of caenorhabditis , pristionchus , drosophila , or tribolium . however , even distant model organisms might be helpful , because the proteins of c. elegans and c. briggsae are different from each other as those of humans and mice ( stein et al . thus , information about tribolium might aid in the development of more distant beetles as genetic model organisms , and zebrafish might be used for other fishes . however , there are some limits . for example , xenopus laevis is polyploid , so it would be unsuitable for many genetic experiments . by contrast , talens work in the diploid frog x. tropicalis ( lei et al . 2013 ) , so these techniques might facilitate its development as a model system . for each new species , the critical steps are as follows : 1 ) sequence the genomes of at least two inbred wild - type strains and identify snps ; 2 ) develop a genetic map using a technique like advanced intercross recombinant inbred lines ( darvasi and soller 1995 ) ; 3 ) use talens to create sets of genetic markers for each chromosome . related species should provide a reliable guide , because most genes with nonsense mutations in c. elegans produced similar phenotypes when knocked out in c. briggsae ; and 4 ) knock out additional genes to make genetic tools . some tools would be determined by technical considerations , such as nmd mutations for analyzing truncations . some would be determined by species , such as him mutants for work with hermaphroditic nematodes , and others would be determined by the biological problem . these steps are now rapid and inexpensive , usually less than $ 80 per gene for reagents . opening up new species for sophisticated genetic analyses should revolutionize evolutionary developmental biology , because the use of null mutants provides a reliable way to compare gene function across species and nonbiased screens give a method for identifying evolutionary novelties ( guo et al . caenorhabditis briggsae mutants were derived from the wild isolate af16 ( fodor et al . 1983 ) and c. sp . two - factor mapping was done as described by brenner ( 1974 ) . from dpy-8 unc-7/++ mothers , we saw 448 wild type , 19 dpy , 22 unc , and 131 dpy unc progeny . from dpy-11 unc-51/++ , we observed 485 wild type , 96 dpy , 105 unc , and 72 dpy uncs . from unc-1 dpy-8/++ , we observed 740 wild type , 63 dpy , 62 unc , and 333 dpy uncs , and from unc-34 dpy-11/++ , we observed 1,008 wild type , 54 dpy , 53 unc , and 249 dpy uncs . first , pairs of custom talens were designed with tale - nt 2.0 software ( https://tale-nt.cac.cornell.edu/ [ last accessed november 19 , 2013 ] ; doyle et al . each target sequence was 1520 nt long and tested by blast to avoid repetitive regions . second , each set of talen repeats was built using a golden gate assembly protocol ( cermak et al . plasmids for the initial steps were purchased from addgene ( http://www.addgene.org/taleffector/goldengatev2/ , last accessed november 19 , 2013 ) . in the final step s1 , supplementary material online ) , rather than into one of the ptal1 - 4 backbone vectors ( cermak et al . 2011 ) . ( 2011 ) with golden gate cloning sites and is optimized for use in caenorhabditis nematodes . third , each plasmid was linearized by digestion with hindiii , treated with 100 g / ml proteinase k and 0.5% sodium dodecyl sulfate ( sds ) for 30 min at 50 c , and purified on a qiaquick column ( qiagen ) . we then synthesized mrna using the sp6 mmessage machine ( ambion ) and purified it on megaclear columns ( ambion ) . we precipitated each mrna and dissolved it in water to give a final concentration of 46 g/l . because pre189 contains a transcribed polya tail of 30 nt , we did not further polyadenylate the messages . fourth , the talen mrnas were combined to produce a solution that was 23 g/l for each message . after injection , animals were soaked in recovery buffer ( evans 2006 ) and picked onto individual plates . at 20 c , the f1 progeny from a 6- to 32-h time window following the injection was singled to new plates , and f2 animals that carried mutations were identified by phenotype or by pcr analysis of the target site . generally , we amplified fragments that were 60100 nt long and separated them by size on 10% polyacrylamide gels ( fig . more subtle changes were detected by the loss of a restriction site or the use of cel1 nuclease ( wood et al . caenorhabditis briggsae mutants were derived from the wild isolate af16 ( fodor et al . 1983 ) and c. sp . two - factor mapping was done as described by brenner ( 1974 ) . from dpy-8 unc-7/++ mothers , we saw 448 wild type , 19 dpy , 22 unc , and 131 dpy unc progeny . from dpy-11 unc-51/++ , we observed 485 wild type , 96 dpy , 105 unc , and 72 dpy uncs . from unc-1 dpy-8/++ , we observed 740 wild type , 63 dpy , 62 unc , and 333 dpy uncs , and from unc-34 dpy-11/++ , we observed 1,008 wild type , 54 dpy , 53 unc , and 249 dpy uncs . first , pairs of custom talens were designed with tale - nt 2.0 software ( https://tale-nt.cac.cornell.edu/ [ last accessed november 19 , 2013 ] ; doyle et al . each target sequence was 1520 nt long and tested by blast to avoid repetitive regions . second , each set of talen repeats was built using a golden gate assembly protocol ( cermak et al . plasmids for the initial steps were purchased from addgene ( http://www.addgene.org/taleffector/goldengatev2/ , last accessed november 19 , 2013 ) . in the final step , the repeat sequences were cloned into the destination vector pre189 ( supplementary fig . s1 , supplementary material online ) , rather than into one of the ptal1 - 4 backbone vectors ( cermak et al . 2011 ) . ( 2011 ) with golden gate cloning sites and is optimized for use in caenorhabditis nematodes . third , each plasmid was linearized by digestion with hindiii , treated with 100 g / ml proteinase k and 0.5% sodium dodecyl sulfate ( sds ) for 30 min at 50 c , and purified on a qiaquick column ( qiagen ) . we then synthesized mrna using the sp6 mmessage machine ( ambion ) and purified it on megaclear columns ( ambion ) . we precipitated each mrna and dissolved it in water to give a final concentration of 46 g/l . because pre189 contains a transcribed polya tail of 30 nt , we did not further polyadenylate the messages . fourth , the talen mrnas were combined to produce a solution that was 23 g/l for each message . after injection , animals were soaked in recovery buffer ( evans 2006 ) and picked onto individual plates . at 20 c , the f1 progeny from a 6- to 32-h time window following the injection was singled to new plates , and f2 animals that carried mutations were identified by phenotype or by pcr analysis of the target site . generally , we amplified fragments that were 60100 nt long and separated them by size on 10% polyacrylamide gels ( fig . more subtle changes were detected by the loss of a restriction site or the use of cel1 nuclease ( wood et al .
although evolutionary studies of gene function often rely on rna interference , the ideal approach would use reverse genetics to create null mutations for cross - species comparisons and forward genetics to identify novel genes in each species . 
 we have used transcription activator - like effector nucleases ( talens ) to facilitate both approaches in caenorhabditis nematodes . 
 first , by combining golden gate cloning and talen technology , we can induce frameshifting mutations in any gene . 
 second , by combining this approach with bioinformatics we can predict and create the resources needed for forward genetic analysis in species like caenorhabditis briggsae . although developing genetic model organisms used to require years to isolate marker mutations , balancers , and tools , with talens , these reagents can now be produced in months . 
 furthermore , the analysis of nonsense mutants in related model organisms allows a directed approach for making these markers and tools . when used together , these methods could simplify the adaptation of other organisms for forward and reverse genetics .