Methods and compositions for assessing crispr/cas-mediated disruption or excision and crispr/cas-induced recombination with an exogenous donor nucleic acid in vivo

Abstract

Methods and compositions are provided for assessing CRISPR/Cas-mediated non-homologous end joining (NHEJ) activity and/or CRISPR/Cas-induced recombination of a target genomic locus with an exogenous donor nucleic acid in vivo or ex vivo. The methods and compositions employ non-human animals comprising a CRISPR reporter such as a genomically integrated CRISPR reporter for detecting and measuring targeted excision of a sequence between two CRISPR/Cas nuclease cleavage sites or disruption of a sequence near a CRISPR/Cas nuclease cleavage site and/or measuring CRISPR/Cas-induced recombination of the CRISPR reporter with an exogenous donor nucleic acid to convert the coding sequence for a first reporter protein to the coding sequence for a different second reporter protein. Methods and compositions are also provided for making and using these non-human animals.

1 . A method of testing the ability of a CRISPR/Cas nuclease to excise a genomic nucleic acid in vivo, comprising:
 (a) introducing into a non-human animal comprising a CRISPR reporter for assessing CRISPR/Cas-induced excision of a nucleic acid between first and second guide RNA target sequences:
 (i) a first guide RNA or a DNA encoding the first guide RNA, wherein the first guide RNA is designed to hybridize to the first guide RNA target sequence in the CRISPR reporter; 
 (ii) a second guide RNA or a DNA encoding the second guide RNA, wherein the second guide RNA is designed to hybridize to the second guide RNA target sequence in the CRISPR reporter; and 
 (iii) a Cas protein or a nucleic acid encoding the Cas protein; 
 wherein the CRISPR reporter is integrated at a target genomic locus and comprises a first polyadenylation signal flanked by the first and second guide RNA target sequences followed by a reporter cassette comprising a coding sequence for a first reporter protein and a coding sequence for a second reporter protein in any order, 
 wherein the first reporter protein and the second reporter protein are different; and 
   (b) measuring the activity or expression of at least one of the first and second reporter proteins.   
     
     
         2 . The method of  claim 1 , wherein one of the first and second reporter proteins comprises a fluorescent reporter protein. 
     
     
         3 . The method of  claim 2 , wherein the fluorescent reporter protein comprises an enhanced green fluorescent protein (eGFP) or an enhanced blue fluorescent protein (eBFP). 
     
     
         4 . The method of  claim 2 , wherein the first and second reporter proteins comprise the fluorescent reporter protein and a non-fluorescent reporter protein. 
     
     
         5 . The method of  claim 4 , wherein the fluorescent reporter protein can be detected in a flow cytometry assay, and the non-fluorescent protein can be detected in a histochemical assay. 
     
     
         6 . The method of  claim 1 , wherein one of the first and second reporter proteins comprises a beta-galactosidase protein. 
     
     
         7 . The method of  claim 1 , wherein the first polyadenylation signal is flanked by recombinase recognition sites for a first recombinase. 
     
     
         8 . The method of  claim 7 , wherein the recombinase recognition sites for the first recombinase are loxP sequences. 
     
     
         9 . The method of  claim 1 , wherein the reporter cassette comprises a multicistronic nucleic acid comprising the coding sequence for the first reporter protein and the coding sequence for the second reporter protein separated by an intervening internal ribosome entry site (IRES) or an intervening 2A peptide coding sequence. 
     
     
         10 . The method of  claim 9 , wherein the multicistronic nucleic acid comprises a beta-galactosidase coding sequence and an enhanced blue fluorescent protein (eBFP) coding sequence or an enhanced green fluorescent protein (eGFP) coding sequence separated by an intervening P2A peptide coding sequence. 
     
     
         11 . The method of  claim 1 , wherein the CRISPR reporter is operably linked to an endogenous promoter at the target genomic locus. 
     
     
         12 . The method of  claim 1 , wherein the 5′ end of the CRISPR reporter comprises a 3′ splicing sequence. 
     
     
         13 . The method of  claim 1 , wherein the CRISPR reporter further comprises a selection cassette. 
     
     
         14 . The method of  claim 13 , wherein the selection cassette is flanked by recombinase recognition sites for a second recombinase. 
     
     
         15 . The method of  claim 13 , wherein the selection cassette comprises a drug resistance gene. 
     
     
         16 . The method of  claim 1 , wherein the distance between the first guide RNA target sequence and the second guide RNA target sequence is less than 500 base pairs. 
     
     
         17 . The method of  claim 1 , wherein the first guide RNA target sequence and the second guide RNA target sequence are identical, and each comprises SEQ ID NO: 41. 
     
     
         18 . The method of  claim 1 , wherein the non-human animal is a mouse or a rat. 
     
     
         19 . The method of  claim 1 , wherein the non-human animal is a rat. 
     
     
         20 . The method of  claim 1 , wherein the non-human animal is a mouse. 
     
     
         21 . The method of  claim 1 , wherein the target genomic locus is a safe harbor locus. 
     
     
         22 . The method of  claim 21 , wherein the safe harbor locus is a Rosa26 locus. 
     
     
         23 . The method of  claim 22 , wherein the CRISPR reporter is inserted into the first intron of the Rosa26 locus. 
     
     
         24 . The method of  claim 1 , wherein the non-human animal is a mouse,
 wherein the target genomic locus is a Rosa26 locus, and   wherein the CRISPR reporter is operably linked to an endogenous Rosa26 promoter, is inserted into the first intron of the Rosa26 locus, and comprises from 5′ to 3′:   (a) a 3′ splicing sequence;   (b) a first polyadenylation signal flanked by:
 (i) first and second loxP sites; and 
 (ii) first and second guide RNA target sequences, wherein the first guide RNA target sequence and the second guide RNA target sequence are identical, and each comprises SEQ ID NO: 41; and 
   (c) a reporter cassette, comprising from 5′ to 3′:
 (i) a beta-galactosidase coding sequence; 
 (ii) a P2A coding sequence; 
 (iii) an enhanced blue fluorescent protein (eBFP) coding sequence, wherein the eBFP coding sequence comprises a third guide RNA target sequence comprising SEQ ID NO: 42; and 
 (iv) a second polyadenylation signal, wherein the first polyadenylation signal and the second polyadenylation signal are different. 
   
     
     
         25 . The method of  claim 24 , wherein the CRISPR reporter further comprises:
 (d) a selection cassette 3′ of the reporter cassette, wherein the selection cassette is flanked by FRT sites and comprises from 5′ to 3′:
 (i) a neomycin phosphotransferase coding sequence operably linked to a human ubiquitin promoter; and 
 (ii) a third polyadenylation signal. 
   
     
     
         26 . The method of  claim 1 , wherein the non-human animal is heterozygous for the CRISPR reporter at the target genomic locus. 
     
     
         27 . The method of  claim 1 , wherein the non-human animal is homozygous for the CRISPR reporter at the target genomic locus. 
     
     
         28 . The method of  claim 1 , wherein the Cas protein is a Cas9 protein. 
     
     
         29 . The method of  claim 1 , wherein step (a) comprises introducing the Cas protein into the non-human animal. 
     
     
         30 . The method of  claim 1 , wherein step (a) comprising introducing the nucleic acid encoding the Cas protein into the non-human animal, wherein the nucleic acid encoding the Cas protein is a messenger RNA encoding the Cas protein. 
     
     
         31 . The method of  claim 1 , wherein step (a) comprises introducing the nucleic acid encoding the Cas protein into the non-human animal, wherein the nucleic acid encoding the Cas protein is a DNA encoding the Cas protein, wherein the DNA is operably linked to a promoter active in one or more cell types in the non-human animal. 
     
     
         32 . The method of  claim 1 , wherein the first guide RNA and the second guide RNA are identical, and each comprises the sequence set forth in SEQ ID NO: 2. 
     
     
         33 . The method of  claim 1 , wherein step (a) comprises introducing the first guide RNA and the second guide RNA into the non-human animal. 
     
     
         34 . The method of  claim 1 , wherein step (a) comprises introducing the DNA encoding first guide RNA and the DNA encoding the second guide RNA into the non-human animal, wherein the DNA encoding the first guide RNA and the DNA encoding the second guide RNA are each operably linked to a promoter active in one or more cell types in the non-human animal. 
     
     
         35 . The method of  claim 1 , wherein the introducing in step (a) comprises adeno-associated virus (AAV)-mediated delivery, lipid nanoparticle-mediated delivery, or hydrodynamic delivery. 
     
     
         36 . The method of  claim 35 , wherein the introducing in step (a) comprises AAV-mediated delivery. 
     
     
         37 . The method of  claim 36 , wherein the introducing in step (a) comprises AAV8-mediated delivery, and step (b) comprises measuring activity of the at least one of the first and second reporter proteins in the liver of the non-human animal. 
     
     
         38 . The method of  claim 1 , wherein the at least one of the first and second reporter proteins measured in step (b) is a fluorescent reporter protein, and step (b) comprises a flow cytometry assay. 
     
     
         39 . The method of  claim 1 , wherein the at least one of the first and second reporter proteins measured in step (b) is a beta-galactosidase protein, and step (b) comprises a histochemical staining assay. 
     
     
         40 . A method of optimizing the ability of a CRISPR/Cas nuclease to excise a genomic nucleic acid in vivo, comprising:
 (I) performing the method of  claim 1  a first time in a first non-human animal;   (II) changing a variable and performing the method of step (I) a second time with the changed variable in a second non-human animal; and   (III) comparing the activity or expression of the at least one of the first and second reporter proteins in step (I) with the activity or expression of the at least one of the first and second reporter proteins in step (II), and selecting the method resulting in the higher activity or expression of the at least one of the first and second reporter proteins.