Multifunctional Alleles

Abstract

Nucleic acid constructs and methods for rendering modifications to a genome are provided, wherein the modifications comprise null alleles, conditional alleles and null alleles comprising COINs. Multifunctional alleles (MFA) are provided, as well as methods for making them, which afford the ability in a single targeting to introduce an allele that can be used to generate a null allele, a conditional allele, or an allele that is a null allele and that further includes a COIN. MFAs comprise pairs of cognate recombinase recognition sites, an actuating sequence and/or a drug selection cassette, and a nucleotide sequence of interest, and a COIN, wherein upon action of a recombinase a conditional allele with a COIN is formed. In a further embodiment, action of a second recombinase forms an allele that contains only a COIN in sense orientation. In a further embodiment, action by a third recombinase forms an allele that contains only the actuating sequence in sense orientation.

1 . A nucleic acid construct, comprising:
 (a) targeting arms for directing the nucleic acid construct to a target gene of a nucleic acid of a cell;   (b) an actuating sequence in sense orientation with respect to transcription of the target gene, and a drug selection cassette (DSC) in sense or antisense orientation;   (b) in antisense orientation a nucleotide sequence of interest (NSI) and a COIN; and,   (c) recombinable units that recombine upon exposure to a first recombinase to form a conditional allele that
 (i) lacks the actuating sequence and the DSC, and, 
 (ii) contains the NSI in sense orientation and the COIN in antisense orientation; 
   wherein the recombinable units comprise two first pairs of cognate recombinase recognition sites recognized by the first recombinase, two second pairs of cognate recombinase recognition sites recognized by a second recombinase, and one third pair of cognate recombinase recognitions sites recognized by a third recombinase; wherein the first pairs, the second pairs, and the third pairs of recombinase recognition sites are recognized by different recombinases.   
     
     
         2 . The nucleic acid construct of  claim 1 , wherein the conditional allele when further exposed to a second site-specific recombinase, recombines to form an allele lacking the NSI and having the COIN in sense orientation. 
     
     
         3 . A method for modifying a non-human cell, comprising:
 targeting a nucleotide sequence of interest in a cell with a nucleic acid construct of  claim 1  to form a targeted cell, exposing the targeted cell to the first recombinase to form the conditional allele, and exposing the conditional allele to a second recombinase that excises the NSI and places the COIN in sense orientation.   
     
     
         4 . The method of  claim 3 , wherein the NSI in the cell is an exon or a nucleotide sequence that is associated with a phenotype. 
     
     
         5 . The method of  claim 4 , wherein the nucleotide sequence of interest in the cell is replaced by the nucleic acid construct of  claim 1 . 
     
     
         6 . The method of  claim 5 , wherein the phenotype is determined a first time after forming the targeted cell but before exposure to the first recombinase. 
     
     
         7 . The method of  claim 6 , wherein the phenotype is determined a second time following exposure to the first recombinase. 
     
     
         8 . A nucleic acid construct, comprising:
 a drug selection cassette (DSC), a reporter, a COIN, a nucleotide sequence of interest (NSI), and five pairs of recombinase sites arranged among the reporter, the DSC, the COIN, and the NSI;   wherein no pair of recombinase sites is identical to any other pair, and wherein a first two pairs of recombinase sites are recognized by the same first recombinase, a second two pairs of recombinase sites are recognized by the same second recombinase, and a fifth pair of recombinase sites are recognized by a third recombinase;   wherein the first, second, and third recombinases are not the same, and wherein, with respect to direction of transcription, the reporter is in sense orientation, the NSI is in antisense orientation, the COIN is in antisense orientation, and the DSC is in sense or antisense orientation; and,   wherein the first two pairs and the second two pairs of recombinase sites are arranged such that upon exposure to the first recombinase, a modified allele forms wherein the first two pairs of recombinase sites direct excision of the reporter, excision of the DSC, inversion of the NSI to sense orientation, and the COIN is maintained in antisense orientation, and either (a) the second two pairs of recombinase sites are arranged such that upon exposure to the second recombinase the NSI is excised and the COIN is placed in sense orientation; or (b) the second two pairs of recombinase sites are arranged such that upon exposure to the second recombinase the NSI is placed in antisense orientation and the COIN is placed in sense orientation.   
     
     
         9 . A nucleic acid construct according to  claim 8 , wherein the fifth pair of recombinase sites are arranged such that upon exposure to the third recombinase and in the absence of exposure to the first or the second recombinase, the fifth recombinase excises that COIN, the NSI, and the DSC, and maintains the reporter in the sense orientation. 
     
     
         10 . A nucleic acid construct according to  claim 8 , wherein the nucleic acid construct is inserted into a gene, and with respect to the direction of transcription of the gene, from 5′ to 3′, the construct comprises a reporter in sense orientation, a DSC in sense or antisense orientation, an NSI in antisense orientation, and a COIN in antisense orientation. 
     
     
         11 . A nucleic acid construct according to  claim 10 , wherein the DSC is in sense orientation. 
     
     
         12 . A nucleic acid construct according to  claim 8 , wherein the nucleic acid construct is inserted into a gene, and with respect to the direction of transcription of the gene, from 5′ to 3′, the construct comprises a COIN in antisense orientation, an NSI in antisense orientation, a DSC in sense or antisense orientation, and a reporter in sense orientation. 
     
     
         13 . A nucleic acid construct according to  claim 12 , wherein the DSC is in sense orientation. 
     
     
         14 . A nucleic acid construct according to  claim 8 , wherein the nucleic acid construct is inserted into a gene, and with respect to the direction of transcription of the gene, from 5′ to 3′, the construct comprises an NSI in antisense orientation, a DSC in sense or antisense orientation, a reporter in sense orientation, and a COIN in antisense orientation. 
     
     
         15 . A nucleic acid construct according to  claim 14 , wherein the DSC is in sense orientation. 
     
     
         16 . A nucleic acid construct according to  claim 8 , wherein the nucleic acid construct is inserted into a gene, and with respect to the direction of transcription of the gene, from 5′ to 3′, the construct comprises a reporter in sense orientation, a DSC in sense or antisense orientation, an NSI in antisense orientation, and a COIN in antisense orientation. 
     
     
         17 . A nucleic acid construct according to  claim 16 , wherein the DSC is in sense orientation. 
     
     
         18 . A nucleic acid construct according to  claim 8 , wherein the nucleic acid construct is inserted into a gene, and with respect to the direction of transcription of the gene, from 5′ to 3′, the construct comprises a COIN in antisense orientation, an NSI in antisense orientation, a DSC in sense or antisense orientation, and a reporter in sense orientation. 
     
     
         19 . A nucleic acid construct according to  claim 18 , wherein the DSC is in sense orientation. 
     
     
         20 . A nucleic acid construct according to  claim 8 , wherein the nucleic acid construct is inserted into a gene, and with respect to the direction of transcription of the gene, from 5′ to 3′, the construct comprises an NSI in antisense orientation, a DSC in sense or antisense orientation, a reporter in sense orientation, and a COIN in antisense orientation.