the first core functionality of the laglidadg homing endonuclease database and engineering server ( lahedes ) resource is an updateable collection of wild - type and engineered lhes that have been shown to cleave precisely defined dna sequences corresponding to their target sites in original host gene , and for which the exact positioning and orientation of the target site relative to the lhe s n- and c - terminal domains are known . in the database ,
the target sites are entered and listed as 22-bp sequences that exactly span the center of four consecutive bases ( termed the 
central four target region ) that are converted into mutually cohesive four - base 3-overhangs by all known lhes .
the orientation of the target site relative to the bound endonuclease ( i.e. which half - sites are engaged by the n- and c - terminal domains of the lhe , respectively ) is defined based on biochemical and/or structural analyses . in this convention , the left half - site ( basepairs 11 to 1 )
is contacted by residues from the n - terminal lhe domain , while the right half - site ( basepairs + 1 to + 11 ) is bound by the c - terminal domain .
entry tool for addition of new lhes to the database provides individual fields for the enzyme name , its amino acid sequence ( entered as a fasta character string ) , its dna target site ( entered from basepair 11 to + 11 as described above ) and a notes field for the entry of additional information for the endonuclease .
this information usually includes ( i ) the host organism and corresponding host gene ; ( ii ) the relationship of the lhe to any surrounding self - splicing elements ( introns or inteins ) or its existence as a  stand - alone endonuclease ; ( iii ) links to genomic or structural information for the endonuclease ; and ( iv ) the nature of experiments that have validated the lhe 's activity .
chimeric lhes ( which correspond to various types of structural fusions between n- and c - terminal lhe domains ) ( 2527 ) 
monomerized lhes ( which correspond to homodimeric endonucleases that have been converted into single - chain reagents through the fusion and tethering of two protein subunits with an artificial peptide linker ) ( 28,29 ) and fully redesigned lhes ( for those that have been entirely retargeted to genomic target sites and that have been biochemically characterized at a level that matches their wild - type parental enzyme ) ( 20 ) . in each case , the same general entry tools and note fields are provided , with individualized instructions for the information required for adequate archiving . finally , it is also possible to enter the names , protein sequences and biological origins of  pseudo - endonucleases , which correspond to lhe genes that do not appear to encode stable or active enzymes ( a category motivated by the observation that a significant fraction of homing endonuclease genes have accumulated disabling mutations subsequent to the successful invasion of their host gene ) ( 30 ) . the rules for naming new lhes , following the conventions described in roberts et al .
( 31 ) are explicitly described and followed by the lahedes database , including instructions to examine the rebase database ( 23 ) to ensure that unique acronyms for novel host species and genus are chosen . in general
, the entry tool recommends adding an additional character to the end of the acronym to further denote the source of the lhe in cases where a newly discovered enzyme is derived from a mitochondrial (  m ) or chloroplast (  c ) genome .
certain enzymes that have already enjoyed a long history in the published literature and corresponding databases ( such as i - scei , i - crei , etc . )
as mentioned above , the browser is limited to those lhes that have been sufficiently characterized that they can be utilized for the creation of gene - targeting reagents ( a list that currently spans 24 enzymes ) .
however , the resource is open - ended and available for deposition of any lhes that meet the necessary criterion for protein engineering and selection experiments .
in addition to the exact sequence of their naturally occurring dna targets ( which usually corresponds to the site in the host gene which is cleaved by the homing endonuclease , resulting in invasion of that site by the mobile he gene and surrounding dna sequences ) , the specificity of a given lhe in many cases is also represented in the database by an additional  position  weight  matrix ( pwm ) ( 32 ) .
this pwm takes into account any positions in the dna target where base substitutions are tolerated in cleavage experiments ( in other words , positions in the dna target that are recognized with reduced fidelity ) .
this feature of lhes ( the ability to tolerate individual polymorphisms in their natural target sites ) is an evolutionary consequence of the need to accommodate the natural drift in the sequence of their host gene targets , and to effectively execute ectopic transfers into new host genes that contain related target sites when the opportunity arises ( 30,33 ) . in particular , lhe reading frames that are found within protein - coding genes
often tend to display reduced fidelity at positions corresponding to  wobble positions in the host gene reading frame ( 33 ) [ a feature of dna recognition that is also explicitly accounted for in the homebase webserver ( 21 ) ] . the use of an experimentally determined pwm as a search - scoring matrix exploits this property , but reduces the risk of false positives that might arise from enzymes that depart from this general rule for recognition specificity .
dna recognition is important in enzyme redesign experiments , where mismatches between the wild - type target site and the desired dna target which correspond to well - tolerated polymorphisms can be accommodated with minimal effort during the engineering or selection process .
accounting for the specificity profile of an lhe is also important in predicting potential genomic  off - target sites , as well as regions of the protein  dna interface where specificity may be enhanced through additional engineering .
pwms are entered into the database with a separate entry tool , using information derived either from direct biochemical measurements of the enzyme 's ability to cleave alternative target site variants ( 34 ) or from experiments that identify collections of cleavable dna target variants from pools of partially randomized sites ( 33 ) . in either case , the relative ability of the enzyme to accommodate each possible base at each possible position in the target site is usually entered using a scale of 1.0 ( wild - type cleavage activity or recovery for a given base ) to 0.0 ( complete lack of cleavage activity or recovery for a given base ) .
the resulting pwm values are displayed by the database both as a sequence logo plot , and as raw text that can be incorporated into separate computational analyses ( figure 1 ) . 

specificity profiles , reported as pwms that have been experimentally determined for a number of lhes can be entered individually and are available on the site as part of the browser functionality .
these matrices are displayed in graphical format as a sequence logo plot ( left ) and in tabular format .
the ability of the enzyme to cleave dna targets that contain a given nucleotide at a specific position within the target site is indicated by letter height , and positions with greater overall information content are more specifically recognized .
the profile displayed in this figure was determined in a series of biochemical experiments where the cleavability of target site variants was individually measured ( 34 ) . the specificity profile ( pwm ) for the i - anii lhe .
specificity profiles , reported as pwms that have been experimentally determined for a number of lhes can be entered individually and are available on the site as part of the browser functionality .
these matrices are displayed in graphical format as a sequence logo plot ( left ) and in tabular format .
the ability of the enzyme to cleave dna targets that contain a given nucleotide at a specific position within the target site is indicated by letter height , and positions with greater overall information content are more specifically recognized . the profile displayed in this figure
was determined in a series of biochemical experiments where the cleavability of target site variants was individually measured ( 34 ) .
in addition to target sites and pwms , the database accommodates lists of residues for each homing endonuclease that might be potentially useful in any redesign or selection process , and links the information within those lists to the output of target site searches ( described below ) .
after entry of a new wild - type lhe , the entry tool for  contact modules is then populated by that enzyme in a drop - down menu that allows an investigator to list those amino acids that the investigator believes to be within contact distance to individual clusters of target sites basepairs .
modules across the target site , that can be incorporated into experiments to alter the enzyme 's recognition specificity within that same dna module .
for example , the i - onui endonuclease displays direct and water - mediated contacts between the 11 to 9  bp positions and at least seven separate amino acid side - chains : asn 32 , lys 34 , ser 35 , ser 36 , val 37 , gly 38 and ser 40 ( 20 ) ; all are listed as residues that comprise the  dna contact module for those three corresponding basepairs .
the choice of amino acids that should populate individual dna contact modules can be derived either from crystallographic structures of a corresponding lhe 
the contact residues entered into the database for the i - crei homodimeric lhe and the i - onui monomeric lhe have been extensively validated for engineering purposes , and those can be used as an initial reference for the entry of corresponding residue lists for homologous lhes .
the premise behind entering amino acid contact lists in a modular format is based on past experimental observations that sequence recognition within the lhe 
dna interface often displays considerable context dependence and cross - talk between adjacent positions ( 28,34,35 ) , such that even when only 1-bp substitution is required in the novel target site , best practice often dictates that residues involved in contacts to the 
n  +  1 basepairs should also be subjected to redesign or selection . for many enzymes in the database , specific mutations in the dna contacting residues described above
have been shown to be associated with defined alterations in target sequence specificity ( 20,34,36 ) .
for example , incorporation of three amino acid substitutions in the monomerized version of i - msoi ( i30e , s43r andi85y ) results in a shift in specificity from a wild - type a : t basepair at position 8 ( termed  8a in the database ) to preferential recognition of a g : c basepair at the same position (  8g ) ( 35 ) .
in addition to the entry tool for wild - type dna contacting residues , an additional  specificity changing mutation entry tool is provided that allows the archiving of such substitutions in the protein scaffold and the corresponding change in dna base preference in the altered target site .
similar to the list of wild - type dna contacting residues , the identities of such mutations are provided to the user both in the endonuclease browser tool , and as a subsequent link from the output of the genomic sequence target site searches to aid in the design of engineering or selection experiments .
the first core functionality of the laglidadg homing endonuclease database and engineering server ( lahedes ) resource is an updateable collection of wild - type and engineered lhes that have been shown to cleave precisely defined dna sequences corresponding to their target sites in original host gene , and for which the exact positioning and orientation of the target site relative to the lhe s n- and c - terminal domains are known . in the database ,
the target sites are entered and listed as 22-bp sequences that exactly span the center of four consecutive bases ( termed the 
central four target region ) that are converted into mutually cohesive four - base 3-overhangs by all known lhes .
the orientation of the target site relative to the bound endonuclease ( i.e. which half - sites are engaged by the n- and c - terminal domains of the lhe , respectively ) is defined based on biochemical and/or structural analyses . in this convention , the left half - site ( basepairs 11 to 1 )
is contacted by residues from the n - terminal lhe domain , while the right half - site ( basepairs + 1 to + 11 ) is bound by the c - terminal domain .
entry tool for addition of new lhes to the database provides individual fields for the enzyme name , its amino acid sequence ( entered as a fasta character string ) , its dna target site ( entered from basepair 11 to + 11 as described above ) and a notes field for the entry of additional information for the endonuclease .
this information usually includes ( i ) the host organism and corresponding host gene ; ( ii ) the relationship of the lhe to any surrounding self - splicing elements ( introns or inteins ) or its existence as a  stand - alone endonuclease ; ( iii ) links to genomic or structural information for the endonuclease ; and ( iv ) the nature of experiments that have validated the lhe 's activity .
chimeric lhes ( which correspond to various types of structural fusions between n- and c - terminal lhe domains ) ( 2527 ) 
monomerized lhes ( which correspond to homodimeric endonucleases that have been converted into single - chain reagents through the fusion and tethering of two protein subunits with an artificial peptide linker ) ( 28,29 ) and fully redesigned lhes ( for those that have been entirely retargeted to genomic target sites and that have been biochemically characterized at a level that matches their wild - type parental enzyme ) ( 20 ) . in each case , the same general entry tools and note fields are provided , with individualized instructions for the information required for adequate archiving . finally , it is also possible to enter the names , protein sequences and biological origins of  pseudo - endonucleases , which correspond to lhe genes that do not appear to encode stable or active enzymes ( a category motivated by the observation that a significant fraction of homing endonuclease genes have accumulated disabling mutations subsequent to the successful invasion of their host gene ) ( 30 ) . the rules for naming new lhes , following the conventions described in roberts et al .
( 31 ) are explicitly described and followed by the lahedes database , including instructions to examine the rebase database ( 23 ) to ensure that unique acronyms for novel host species and genus are chosen . in general
, the entry tool recommends adding an additional character to the end of the acronym to further denote the source of the lhe in cases where a newly discovered enzyme is derived from a mitochondrial (  m ) or chloroplast (  c ) genome .
certain enzymes that have already enjoyed a long history in the published literature and corresponding databases ( such as i - scei , i - crei , etc . )
as mentioned above , the browser is limited to those lhes that have been sufficiently characterized that they can be utilized for the creation of gene - targeting reagents ( a list that currently spans 24 enzymes ) .
however , the resource is open - ended and available for deposition of any lhes that meet the necessary criterion for protein engineering and selection experiments .
in addition to the exact sequence of their naturally occurring dna targets ( which usually corresponds to the site in the host gene which is cleaved by the homing endonuclease , resulting in invasion of that site by the mobile he gene and surrounding dna sequences ) , the specificity of a given lhe in many cases is also represented in the database by an additional  position  weight  matrix ( pwm ) ( 32 ) .
this pwm takes into account any positions in the dna target where base substitutions are tolerated in cleavage experiments ( in other words , positions in the dna target that are recognized with reduced fidelity ) .
this feature of lhes ( the ability to tolerate individual polymorphisms in their natural target sites ) is an evolutionary consequence of the need to accommodate the natural drift in the sequence of their host gene targets , and to effectively execute ectopic transfers into new host genes that contain related target sites when the opportunity arises ( 30,33 ) .
in particular , lhe reading frames that are found within protein - coding genes often tend to display reduced fidelity at positions corresponding to  wobble positions in the host gene reading frame ( 33 ) [ a feature of dna recognition that is also explicitly accounted for in the homebase webserver ( 21 ) ] .
the use of an experimentally determined pwm as a search - scoring matrix exploits this property , but reduces the risk of false positives that might arise from enzymes that depart from this general rule for recognition specificity . the need to account for relaxed fidelity in lhe 
dna recognition is important in enzyme redesign experiments , where mismatches between the wild - type target site and the desired dna target which correspond to well - tolerated polymorphisms can be accommodated with minimal effort during the engineering or selection process .
accounting for the specificity profile of an lhe is also important in predicting potential genomic  off - target sites , as well as regions of the protein  dna interface where specificity may be enhanced through additional engineering .
pwms are entered into the database with a separate entry tool , using information derived either from direct biochemical measurements of the enzyme 's ability to cleave alternative target site variants ( 34 ) or from experiments that identify collections of cleavable dna target variants from pools of partially randomized sites ( 33 ) . in either case , the relative ability of the enzyme to accommodate each possible base at each possible position in the target site is usually entered using a scale of 1.0 ( wild - type cleavage activity or recovery for a given base ) to 0.0 ( complete lack of cleavage activity or recovery for a given base ) .
the resulting pwm values are displayed by the database both as a sequence logo plot , and as raw text that can be incorporated into separate computational analyses ( figure 1 ) . 

figure 1.the specificity profile ( pwm ) for the i - anii lhe . specificity profiles , reported as pwms that have been experimentally determined for a number of lhes can be entered individually and are available on the site as part of the browser functionality .
these matrices are displayed in graphical format as a sequence logo plot ( left ) and in tabular format .
the ability of the enzyme to cleave dna targets that contain a given nucleotide at a specific position within the target site is indicated by letter height , and positions with greater overall information content are more specifically recognized . the profile displayed in this figure
was determined in a series of biochemical experiments where the cleavability of target site variants was individually measured ( 34 ) . the specificity profile ( pwm ) for the i - anii lhe .
specificity profiles , reported as pwms that have been experimentally determined for a number of lhes can be entered individually and are available on the site as part of the browser functionality .
these matrices are displayed in graphical format as a sequence logo plot ( left ) and in tabular format .
the ability of the enzyme to cleave dna targets that contain a given nucleotide at a specific position within the target site is indicated by letter height , and positions with greater overall information content are more specifically recognized . the profile displayed in this figure
was determined in a series of biochemical experiments where the cleavability of target site variants was individually measured ( 34 ) .
in addition to target sites and pwms , the database accommodates lists of residues for each homing endonuclease that might be potentially useful in any redesign or selection process , and links the information within those lists to the output of target site searches ( described below ) . after entry of a new wild - type lhe , the entry tool for  contact modules
is then populated by that enzyme in a drop - down menu that allows an investigator to list those amino acids that the investigator believes to be within contact distance to individual clusters of target sites basepairs .
modules across the target site , that can be incorporated into experiments to alter the enzyme 's recognition specificity within that same dna module .
for example , the i - onui endonuclease displays direct and water - mediated contacts between the 11 to 9  bp positions and at least seven separate amino acid side - chains : asn 32 , lys 34 , ser 35 , ser 36 , val 37 , gly 38 and ser 40 ( 20 ) ; all are listed as residues that comprise the  dna contact module for those three corresponding basepairs .
the choice of amino acids that should populate individual dna contact modules can be derived either from crystallographic structures of a corresponding lhe  dna complex , or from homology models constructed from such available structures .
the contact residues entered into the database for the i - crei homodimeric lhe and the i - onui monomeric lhe have been extensively validated for engineering purposes , and those can be used as an initial reference for the entry of corresponding residue lists for homologous lhes .
the premise behind entering amino acid contact lists in a modular format is based on past experimental observations that sequence recognition within the lhe 
dna interface often displays considerable context dependence and cross - talk between adjacent positions ( 28,34,35 ) , such that even when only 1-bp substitution is required in the novel target site , best practice often dictates that residues involved in contacts to the 
n  +  1 basepairs should also be subjected to redesign or selection . for many enzymes in the database , specific mutations in the dna contacting residues described above
have been shown to be associated with defined alterations in target sequence specificity ( 20,34,36 ) .
for example , incorporation of three amino acid substitutions in the monomerized version of i - msoi ( i30e , s43r andi85y ) results in a shift in specificity from a wild - type a : t basepair at position 8 ( termed  8a in the database ) to preferential recognition of a g : c basepair at the same position (  8g ) ( 35 ) .
in addition to the entry tool for wild - type dna contacting residues , an additional  specificity changing mutation entry tool is provided that allows the archiving of such substitutions in the protein scaffold and the corresponding change in dna base preference in the altered target site .
similar to the list of wild - type dna contacting residues , the identities of such mutations are provided to the user both in the endonuclease browser tool , and as a subsequent link from the output of the genomic sequence target site searches to aid in the design of engineering or selection experiments .
the second core functionality of the lahedes resource is the ability to search individual genes or collections of genes for potential matches against lhe target sites based on four separate search criteria : 
 searches for dna sequences that exhibit identity across the central 4  bp of an lhe target site , because those bases are usually not in direct contact with amino acid side chains .
therefore , specificity changes at these basepairs are often not easily achieved through protein engineering or selection ( 34 ) .
additional experiments on individual lhe enzymes that further profile the ability of the enzyme to accommodate individual basepair substitutions in the  central four region of the target site expands the number of hits within a genomic query . for those enzymes where such information is available , the
 central four search algorithm takes such polymorphisms into account when returning potential targets and allows such sites to appear on the list of target site hits.searches for highest possible sequence identity ( fewest dna basepair mismatches ) across a 22-bp dna sequence in the genomic query relative to an lhe s entire physiological target site .
some redesign experiments indicate , not surprisingly , that the most effective way to maintain wild - type levels of affinity , activity and overall specificity is to maintain the highest degree of sequence similarity to the wild - type target ( 20).for those lhes that have had their complete specificity profile determined , the user is provided with the opportunity to search and score potential targets using a  specificity profile pwm described above . in this search algorithm ,
the penalty for a mismatch between a protein s natural target site and a potential genomic target is differentially weighted depending on the fidelity of recognition displayed at each dna basepair position .
the difference between the two search strategies described in ( ii ) versus ( iii ) is simple : the use of a simple identity matrix will return those potential targets with the fewest mismatches relative to the enzyme 's wild - type recognition site , while the use of a scoring matrix that accounts for recognition degeneracy would indicate sites that might be more distantly related to the protein 's wild - type recognition site , while nevertheless indicating more tractable gene - targeting sites.the
i - onui lhe has been systematically tested for its ability to be retargeted towards each possible altered base triplet across the entire length of the dna target site , using a high throughput method that combines yeast surface display and flow cytometry ( 37 ) ( j. jarjour , unpublished data ) . for that enzyme , genomic searches are therefore possible using corresponding modular scoring matrices , returning lists of potential target sites to the investigator that might be accessible for modification through a subsequent experimental strategy that makes use of highthroughput selection from pools of enzyme variants that are randomized at corresponding dna - contacting residues .
searches for dna sequences that exhibit identity across the central 4  bp of an lhe target site , because those bases are usually not in direct contact with amino acid side chains .
therefore , specificity changes at these basepairs are often not easily achieved through protein engineering or selection ( 34 ) .
additional experiments on individual lhe enzymes that further profile the ability of the enzyme to accommodate individual basepair substitutions in the
 central four region of the target site expands the number of hits within a genomic query . for those enzymes where such information is available , the
 central four search algorithm takes such polymorphisms into account when returning potential targets and allows such sites to appear on the list of target site hits .
searches for highest possible sequence identity ( fewest dna basepair mismatches ) across a 22-bp dna sequence in the genomic query relative to an lhe s entire physiological target site .
some redesign experiments indicate , not surprisingly , that the most effective way to maintain wild - type levels of affinity , activity and overall specificity is to maintain the highest degree of sequence similarity to the wild - type target ( 20 ) . for those lhes that have had their complete specificity profile determined ,
the user is provided with the opportunity to search and score potential targets using a  specificity profile pwm described above . in this search algorithm ,
the penalty for a mismatch between a protein s natural target site and a potential genomic target is differentially weighted depending on the fidelity of recognition displayed at each dna basepair position .
the difference between the two search strategies described in ( ii ) versus ( iii ) is simple : the use of a simple identity matrix will return those potential targets with the fewest mismatches relative to the enzyme 's wild - type recognition site , while the use of a scoring matrix that accounts for recognition degeneracy would indicate sites that might be more distantly related to the protein 's wild - type recognition site , while nevertheless indicating more tractable gene - targeting sites .
the i - onui lhe has been systematically tested for its ability to be retargeted towards each possible altered base triplet across the entire length of the dna target site , using a high throughput method that combines yeast surface display and flow cytometry ( 37 ) ( j. jarjour , unpublished data ) . for that enzyme
, genomic searches are therefore possible using corresponding modular scoring matrices , returning lists of potential target sites to the investigator that might be accessible for modification through a subsequent experimental strategy that makes use of highthroughput selection from pools of enzyme variants that are randomized at corresponding dna - contacting residues .
the search algorithm also allows the user to search for genomic sequences that might be targeted through the application of validated 
chimeric homing endonucleases ( where n- and c - terminal domains or dna contacting surfaces of unrelated lhes have been fused or individually modified to create scaffolds that can recognize corresponding chimeric dna target sites ) ( 2527 ) . following genomic target site searches using any of the four methods outlined above ( along with user - chosen endonucleases for the search that can span all or a smaller subset of those lhes that are available in the database ) , a top - scoring list of putative target sequences is returned in a graphical user interface , alongside the enzyme 's wild - type target for comparison ( figure 2a ) .
the output , which can be saved to a text file , indicates the position and identity of all basepairs in the potential target that differs from the enzyme 's wild - type recognition site , and provides links from each base in the candidate targets to information that has been entered about amino acids and mutations in the lhe that might influence specificity and redesign at that position . 
 figure 2.genomic search results .
the human monoamine oxidase b ( maob ) gene was searched for potential target sites matches using the i - onui lhe and either the  identity algorithm ( a ) or the  modular engineerability algorithm ( b ) . for both searches ,
a list of best matches is shown , together with their position in the query sequence , orientation ( forward or reverse ) and scores .
identity search ( panel a , top maob sequence ) was targeted for cleavage at the endogenous chromosomal maob locus by an engineered version of i - onui ( 20 ) ( listed as 
the target site hits , mismatches are shown as lower base bases . in the output from a module search , each score is linked to a more detailed view ( c ) illustrating how well each dna module scores using an 
modules are represented by individual bars ; those that score favorably in a given target are colored blue , while those that do not score as favorably are colored red .
the engineerability scoring scale ( 0 to 10 ; 10 is best ) is shown to the right .
clicking on individual bases or modular bars in the search output provides links to lists of amino acids ( d ) and ( if they are available ) mutations in the protein that are associated with recognition at those positions in the target .
the human monoamine oxidase b ( maob ) gene was searched for potential target sites matches using the i - onui lhe and either the 
identity algorithm ( a ) or the  modular engineerability algorithm ( b ) . for both searches ,
a list of best matches is shown , together with their position in the query sequence , orientation ( forward or reverse ) and scores .
identity search ( panel a , top maob sequence ) was targeted for cleavage at the endogenous chromosomal maob locus by an engineered version of i - onui ( 20 ) ( listed as 
the target site hits , mismatches are shown as lower base bases . in the output from a module search , each score is linked to a more detailed view ( c ) illustrating how well each dna module scores using an 
modules are represented by individual bars ; those that score favorably in a given target are colored blue , while those that do not score as favorably are colored red .
nucleotides that directly match the wild type target are indicated by vertical bars . the engineerability scoring scale ( 0 to 10 ; 10 is best ) is shown to the right .
clicking on individual bases or modular bars in the search output provides links to lists of amino acids ( d ) and ( if they are available ) mutations in the protein that are associated with recognition at those positions in the target . while the search output using the first three scoring algorithms (  central 4 match ,  target identity and  pwm search ) is arranged to clearly display the conservation or mismatch at each individual dna basepair , the output for the  modular search displays individual genomic sequences in the form of information regarding the  engineerability of each protein  dna module that harbors one or more basepair changes ( figure 2b ) . as described above ,
the individual regions and basepairs in potential target sites are linked to previously entered information regarding the identity of amino acids and the corresponding mutations that are involved in the recognition of those positions .
the field of genome and gene engineering is currently experiencing a rapid increase in the amount of information and data relating to the creation of gene - targeting proteins derived from the lhe family .
these include : ( i ) the continuing identification of new lhes from worldwide microbial and metagenomic sequencing projects and ( ii ) the generation of a large numbers of engineered lhe variants with altered dna recognition properties .
one manner in which the full potential and impact of this information can be fully exploited is through the development and maintenance of a computational database and search engine for lhe proteins .
although lhes have historically been considerably more difficult to engineer for altered dna recognition and cleavage properties relative to their artificial , modular zfn and talen counterparts , there still exists considerable industrial and academic activity and motivation to reduce or eliminate the technical gulf that separates them from routine application in genome engineering applications .
it is likely that the combination of highthroughput modular screens for altered specificity and the increasing numbers of wild - type and chimeric lhe protein scaffolds , along with the ability to apply deep sequencing methods to resulting pools of highly active enzyme variants , may soon drive a significant reduction in the time and cost of assembling artificial lhes for the life science community .
barry stoddard is a founder , and jordan jarjour is an employee , of pregenen inc . , which is focused on the development and engineering of lhes for gene targeting applications .
 laglidadg homing endonucleases ( lhes ) are dna cleaving enzymes , also termed  
 meganucleases that are employed as gene - targeting reagents . 
 this use of lhes requires that their dna specificity be altered to match sequences in genomic targets . 
 the choice of the most appropriate lhe to target a particular gene is facilitated by the growing number of such enzymes with well - characterized activities and structures .  
 lahedes ( the laglidadg homing endonuclease database and engineering server ) provides both an online archive of lhes with validated dna cleavage specificities and dna - binding interactions , as well as a tool for the identification of dna sequences that might be targeted by various lhes . 
 searches can be performed using four separate scoring algorithms and user - defined choices of lhe scaffolds . 
 the webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments . 
 the webserver is fully open access and can be found at http://homingendonuclease.net . 
LHE DATABASE
Enzyme and target site entry
Specificity profile (position weight matrix) entry
DNA contact module and redesign mutation entry
LHE SEARCH AND ENGINEERING SERVER
CONCLUSIONS
FUNDING