WO2016132122A1 - Assay construct - Google Patents

Abstract

Disclosed herein are systems, constructs, methods and assays for use in assessing, monitoring, quantifying and/or determining the function, expression, activity and/or efficacy of nucleic acid and/or genome editing systems. The constructs and methods may be used to assess, monitor, quantify and/or determine the function, expression, activity and/or efficacy of, for example, a TALEN. The disclosure provides a construct for assessing or determining the expression, activity, function or efficacy of a nucleic acid/genome editing system, said construct comprising a sequence encoding a reporter element and an editable sequence, wherein the editable sequence is contained within the sequence encoding the reporter element.

Description

ASSAY CONSTRUCT

FIELD OF THE INVENTION

The present invention provides novel methods and assays for determining, assessing, evaluating, quantifying or monitoring the function, expression and/or activity of a nucleic acid and/or genome editing system.

BACKGROUND OF THE INVENTION

Genome editing systems such as zinc fingers, Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and Transcription Activator Like Effectors (TALEs) have become powerful tools for transcriptional activation and genome editing^1"5. In particular, CRISPR and TALE technologies utilise relatively simple molecular biology techniques and toolkits are readily available for end users^6"11.

There are several methods used to assess the functional application of genome editing systems. These include, for example, the surveyor assay and episomal gene repair assay^M'— .

It is among the objects of this invention to provide a novel method for rapidly, efficiently and accurately assessing, determining, evaluating, monitoring and/or quantifying the function, expression and/or activity of a genome editing system.

SUMMARY OF THE INVENTION

The present invention provides systems, constructs, methods and assays for use in assessing, monitoring, quantifying and/or determining the function, expression, activity and/or efficacy of nucleic acid and/or genome editing systems. The constructs and methods described herein may be used to assess, monitor, quantify and/or determine the function, expression, activity and/or efficacy of, for example, a TALEN.

In a first aspect, the invention provides a construct for assessing or determining the expression, activity, function or efficacy of a nucleic acid/genome editing system, said construct comprising a sequence encoding a reporter element and an editable sequence, wherein the editable sequence is contained within the sequence encoding the reporter element. The constructs of this invention may be nucleic acid constructs.

Functional expression of the reporter sequence is prevented by the editable sequence and in particular, its placement and location within the sequence encoding the reporter element. In order to achieve functional reporter expression, the construct of the invention must be contacted with a nucleic acid/genome editing system under conditions which permit the nucleic acid/genome editing system to modify or edit the editable sequence such that functional expression of the reporter sequence can occur.

The sequence encoding one or more reporter elements (referred to hereinafter as the "reporter sequence") may encode any element which, when expressed, is detectable by some means. For example, the expression product of the reporter element sequence may be optically detectable.

The reporter sequence may encode a fluorescent or luminescent protein.

For example, the reporter sequence may encode one or more of Green Fluorescent Protein (GFP: or any associated derivatives or variants thereof - for example enhanced (e) GFP), red fluorescent protein, Zeis Green Fluorescent protein, luciferase, beta- galactosidase, beta-glucuronidase and the like.

The reporter sequence may encode a moiety or agent which is mass detectable and/or a moiety or agent which is able to confer or afford a level of compound (for example antibiotic) resistance.

In view of the above, the (nucleic acid) constructs of the first aspect of this invention may comprise a sequence encoding a fluorescent/luminescent protein and an editable sequence, wherein the editable sequence is contained within the sequence encoding the fluorescent/luminescent protein.

The term "nucleic acid editing system" or "genome editing system" may encompass any system which is capable of manipulating, altering and/or modifying a nucleic acid or genomic sequence. The term "system" embraces any compound or compounds which exhibit or possess genome or nucleic acid editing capabilities. A "system" may comprise one or more proteins and thus the term "nucleic acid/genome editing system" may encompass "nucleic acid/genome editing proteins". For example, a "nucleic acid/genome editing system" may take the form of a proteinaceous moiety, for example a peptide, protein or protein fusion. The nucleic acid/genome editing system may comprise an enzyme, for example an endonuclease or recombinase.

For convenience, all nucleic acid and/or genome editing systems compatible with the systems, constructs and methods/assays of this invention shall be collectively referred to as "genome editing systems".

The "manipulation", "modification" and "alteration" of a genomic or nucleic acid sequence includes the restriction (cutting) of a genomic or nucleic acid sequence and/or the insertion, inversion, removal and/or addition of one or more nucleotides therefrom.

As such, genome editing systems which may be assessed and/or analysed using the constructs of this invention may include peptide or protein systems which are capable of cutting ("restricting") genomic and/or nucleic acid sequences.

A genome editing system may, for example, comprise or take the form of an enzyme or endonuclease moiety. For example, the genome editing system may comprise or take the form of a Transcription activator-like effector nuclease (TALEN). The genome editing system may comprise a recombinase, zinc finger, clustered regularly interspaced short ealindromic repeats (CRISPR) or meganuclease moiety or element. For example, the genome editing system may comprise a zinc finger nuclease.

One of skill will appreciate that the genome editing system may exhibit binding specificity and/or affinity for a particular sequence. As such, the editable sequence of the constructs of this invention may comprise a sequence which is specific for (edited by) and/or bound by, one or more genome editing systems. For example, the editable sequence may comprise a sequence which is specific for (edited by) a genome editing system, the activity or function of which is to be assessed and/or determined.

The editable sequence may comprise a genome editing system binding site and a sequence editable by the genome editing system. The editable sequence may comprise, for example one or more restriction site(s), lox p sites, viral derived sequences (such as, for example a 2A peptide or internal ribosome entry sequence (IRES)), stuffer fragments, modified stuffer fragments and the like.

Where the genome editing system is (or comprises) a TALEN, the editable sequence may comprise a sequence which is bound by the TALE component of the TALEN molecule. One of skill will understand that the precise features (sequence) of the editable sequence will vary depending on the nature (for example specificity) of the genome editing system. As stated, in the case of a TALEN molecule, the sequence specific for the genome editing system may comprise a sequence which is bound by the TALE component of the TALE molecule - thus the sequence will depend on the composition and specificity of the TALE molecule.

Where the genome editing system is (or comprises) a Cre recombinase, the editable sequence may comprise a stop codon. For example the sequence may comprise a lox p site and a stuffer fragment which contains a stop codon.

As stated, the editable sequence is contained within the reporter sequence. For example, the sequence encoding the reporter element may be split or separated by the editable sequence.

The editable sequence may be flanked by first and second reporter sequences. For example, the first reporter sequence may encode part of a reporter element and the second reporter sequence may encode another part of the same reporter element. The first reporter sequence may encode an N-terminus section of the reporter element and the second reporter sequence may encode a C-terminus section of the reporter element. Together, the first and second reporter sequences encode a functional reporter element. Where the reporter element is GFP, the first reporter sequence may encode residues 1 -158 and the second reporter sequence may encode residues 159-end.

The first reporter sequence may be fused or joined to the 5' end of the editable sequence and the second reporter sequence may be fused or joined to the 3' end of the editable sequence. The constructs of this invention are designed or organised such that the location of the editable sequence between sequences which encode the reporter element, prevents functional reporter element expression. For example and without wishing to be bound by theory, the location of an editable sequence within a reporter element sequence may result in a frame shift which prevents functional expression of the reporter element. The frame shift may result in the appearance of one or more stop codons downstream of the editable sequence - i.e. within the second reporter element sequence. One of skill will appreciate that the presence of a stop codon within a nucleic acid sequence will prevent complete translation and/or expression. In the case of the constructs of this invention, the creation of a stop codon downstream of the editable sequence and/or within the second reporter sequence (the stop codon appearing because of the frame shift resulting from the insertion of the editable sequence), will prevent expression of a detectable reporter element.

The constructs of this invention may be designed or organised to ensure that the second reporter sequence is out of frame with the first reporter sequence. For example, the editable sequence may be of a suitable size, to ensure that the second reporter sequence is out of frame with the first reporter sequence. This arrangement prevents functional expression of a reporter element by a construct of this invention. The editable sequence may be regarded as a frame shifting insertion - in other words, the insertion of the editable sequence within the sequence encoding the reporter element, causes the first and second sequences which encode the reporter element (and which are located either side of the editable sequence) to be out of frame with one another.

As such, this invention provides a construct for assessing or determining the function, expression, efficacy and/or activity of a genome editing system, said construct comprising first and second reporter sequences which together encode a functional reporter element, and an editable sequence, wherein the editable sequence is located between the reporter sequences and the second reporter sequence is out of frame with the first reporter sequence. In other words, the editable sequence introduces a frameshift and alters the open reading frame of the second reporter element sequence. Thus the first and second reporter sequences are out of frame with one another. Functional expression of the reporter sequence may only be achieved by restoring the correct open reading frame between the first and second reporter element sequences.

The constructs of this invention may be used to assess or determine whether or not a particular genome editing system is (or has been) expressed or is functional or not. Without wishing to be bound by theory, when a genome editing system is brought into contact with a construct of this invention, the genome editing system "edits" the editable sequence of the construct. Thereafter, subsequent repair events may restore the open reading frame between the reporter element sequences. This results in the expression of a detectable reporter signal (the product of the reporter element). As such, the expression and/or detection of a reporter element following contact between a genome editing system and a construct of this invention is indicative of an expressed and/or functional genome editing system. It should be understood that a construct suitable for the assessment of a particular genome editing system will comprise a sequence that is at least editable by the genome editing system to be assessed.

Again, without wishing to be bound by theory, restoration of the reporter sequence reading frame occurs as a consequence of frame shifts resulting from modification (or manipulation or editing) of the editable sequence by the genome editing system and/or through subsequent (i.e. post editing, manipulation or modification via the genome editing system) repair events including, for example non-homologous end joining (NHEJ). One of skill will appreciate that repair events of this type are error prone and may result in nucleotide deletions, insertions and/or inversions. The result of these errors may be a frame shift in the repaired editable sequence which restores the reading frame of the reporter sequences. The subsequent expression of the edited and repaired construct results in the expression of a functional (and detectable) reporter element.

Where the genome editing system comprises an endonuclease (for example a TALEN molecule) a construct of this invention for assessing and/or determining the expression, efficacy, function and/or activity of the endonuclease, may comprise a sequence which is editable by that endonuclease. Upon contact between the construct and the (functional) endonuclease, the editable sequence may be subject to a double stranded break which is repaired by, for example, error prone non-homologous end joining (NHEJ). This results in mutations, for example, nucleotide deletions or insertions, and a frame shift in the editable sequence (of, for example, -1 , -4 or +1 or +4 or -2, -5 or +2 or +5 or any triplet combination of these) which restores the reporter sequence reading frame. For example, the effect of the mutations (insertions, deletions and the like) is to create an open reading frame in which one part (for example the N terminal part) of the reporter sequence can complement (or act in trans with) the other part (for example the C-terminus part). It should be noted that the precise nature and number of the nucleotide (mutations) deletions or insertions required to restore expression (and permit detection) of the reporter element is variable and may depend upon the precise design of the construct. For example, sequencing after contact between a genome editing system and a construct of this invention reveals that the construct may be repaired by a process which involves NHEJ. In one instance, an editable sequence of a construct of this invention was found to have been altered via the deletion of 10 bp (Δ10), the result being that the N-terminus of the reporter sequence (for example a sequence encoding GFP) was placed in frame with (or relative to) the C-terminus sequence part. This led to restoration of reporter (fluorescent) activity. In other instances, the editable sequence was found to have undergone other mutations including, for example, Δ79 +53, Δ91 +1 10 and Δ93 +83, respectively. The effect of these mutations was to remove the stop codons and generate an Open Reading Frame with the C-terminal reporter sequence domain resulting in the formation of an active reporter sequence/element by complementation with N-terminal GFP.

In view of the above, this invention provides a construct for assessing or determining the expression, function, efficacy and/or activity of an endonuclease, said construct comprising first and second fluorescent/luminescent protein sequences which together encode a functional fluorescent/luminescent protein, and a sequence editable by the endonuclease, wherein the sequence editable by the endonuclease is located between the first and second fluorescent/luminescent protein sequences and the second fluorescent/luminescent protein sequence is out of frame with the first fluorescent/luminescent protein sequence.

The construct may comprise a sequence bound by the genome editing system - that sequence may be part of the editable sequence.

The endonuclease may take the form of a TALEN and the first and second reporter sequences may together encode a functional GFP (or variant or derivative thereof: for example eGFP).

The sequence editable by the endonuclease may comprise a sequence which can be restricted or cut by the endonuclease. For example, where the genome editing system is a TALEN comprising a Fok1 endonuclease, the sequence editable by the endonuclease may comprise a sequence which can be restricted or cut by the Fok1 endonuclease.

An example construct of this invention may comprise the following structure:

As noted earlier, reporter sequence 2 is out of frame with reporter sequence 1 .

As such, the present invention provides a construct for assessing or determining the expression, activity, function or efficacy of a nucleic acid/genome editing system, for example a TALEN, said construct comprising a sequence encoding a GFP reporter element and an editable sequence, wherein the editable sequence is contained within the sequence encoding the reporter element and is located immediately downstream, after or adjacent the codon encoding residue 158 of the GFP reporter element.

Without wishing to be bound by theory, it is suggested that fluorescent proteins such as, for example GFP consists of a number of antiparallel beta strands (in the case of GFP there are 1 1 antiparallel beta strands). When modifying sequences which encode these types of protein (for example by the addition of extra sequences) it is advantageous not to interfere with these motifs. For example, when adding additional sequences into sequences which encode fluorescent proteins such as GFP and the like, attempts should be made to avoid interfering with the integrity of the native structure and integrity. The inventors note that the GFP sequence can tolerate insertions between antiparallel beta strands 4/5, antiparallel beta strands 7/8 and antiparallel beta strands 8 and 9. For example, residue 158 of GFP is located between beta 7 and 8. Other fluorescent proteins such as Venus, cyan and BFP are mutated/modified versions of GFP and as such they too will most likely tolerate insertions at positions within their sequence that do not adversely affect the native structure/integrity (in particular, the antiparallel beta strand motifs). Proteins which are modified forms of GFP may tolerate sequence insertions at positions adjacent to (or upstream of) residue 158. Analysis of other fluorescent proteins for use in the constructs and/or methods of this invention shows that they have structures which are similar to those of GFP. As such, provided similar care is taken to respect the antiparallel beta strand motifs, they should function equally well in the constructs and methods of this invention.

Methods which exploit constructs of this invention do not require the use of, for example, donor or companion vectors which provide copies of all or parts of the (unmodified) reporter element sequence. Moreover, the constructs of this invention may not be subjected to double strand break (DSB) homology directed repair (HDR) events. Further, the constructs disclosed herein may not rely (or require) Single Stranded Annealing processes involving the repair of a double stranded break between two repeat sequences. The constructs of this invention comprise a reporter sequence which is split or separated by some form of target or editable sequence. A construct of this invention may not comprise first and second reporter sequences which are essentially two overlapping and nonfunctional parts of a reporter sequence are separated by an editable or target sequence. In other words first reporter sequence may not overlap (i.e. have the same residues as) part of the second reporter sequence. As stated, the constructs of this invention comprise first and second reporter sequences separated by an editable or target sequence - thus, the reporter sequence may not be complete (i.e. it is split or separated by the target/editable sequence) and the target/editable sequence is located or disposed between the first and second reporter sequences. For example, the construct may not comprise an editable or target sequence located downstream of the reporter sequence. For example, the construct may not comprise a stop codon located near to the start codon (ATG) of a complete (i.e. non-split) reporter sequence.

Without wishing to be bound by theory, the inventors have determined that prior art constructs and methods for assessing the expression, function and/or activity of a genome editing system, in particular those that require the use of companion vectors and the like and which depend on double strand break (DSB) homology directed repair (HDR) events, exhibit poor efficiency and yield low reporter signals (for example fluorescence levels of about 1%- 1.3%). In contrast, the constructs and methods of this invention yield much higher fluorescence - for example between about 4% and 10% fluorescence.

An example (complete) GFP nucleic sequence is given as SEQ ID NO: 1 below. Also shown is the translated GFP protein sequence (see: SEQ ID NO: 2).

SEQ ID NOS 1 (nucleic acid) and 2 (amino acid):

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC

M V S K G E E L F T G V V P I L V E L D GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC

G D V N G H K F S V S G E G E G D A T Y GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACC

G K L T L K F I C T T G K L P V P W P T CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAG L V T T L T Y G V Q C F S R Y P D H M K CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC

Q H D F F K S A M P E G Y V Q E R T I F TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG F K D D G N Y K T R A E V K F E G D T L GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC

V N R I E L K G I D F K E D G N I L G H AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC K L E Y N Y N S H N V Y I M A D K Q K N GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC

G I K V N F K I R H N I E D G S V Q L A GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC

D H Y Q Q N T P I G D G P V L L P D N H TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC

Y L S T Q S A L S K D P N E K R D H M V CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

L L E F V T A A G I T L G M D E L Y K -

As such, SEQ ID NO: 1 represents an example reporter sequence for use in a construct of this invention.

Amino acid Q158 is marked in bold and underline text. An editable sequence (for example a genome editing binding site) may be inserted at (e.g. immediately downstream) this position.

As a consequence, SEQ ID NO: 1 may be split into two reporter sequences - a first sequence (SEQ ID NO: 3 below) encoding the N-terminus part of the GFP (residues 1-158) and a second sequence (SEQ ID NO: 4) encoding the C-terminus part of the GFP (residues 159-end).

SEQ ID NO: 3: an example first reporter sequence encoding the N-terminus part of a GFP (nucleotides 1-474 of SEQ ID NO: 1).

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGT AAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGA AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGC GTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG SEQ ID NO: 4: an example second reporter sequence encoding the C-terminus part of a GFP for use in a construct of this invention (nucleotides 475-720 of SEQ ID NO: 1 ) .

AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCA CCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

An example construct of this invention may comprise SEQ ID NO: 5 (below):

SEQ ID NO: 5

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGT AAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGA AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGC GTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGiiiGC GGCCGCAAGCTT^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^¾ illiliillilGGATCCCTCGAGAAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGA

GGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGC TGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCAC ATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

In SEQ ID NO: 5, the first reporter sequence is provided by nucleotides 1 -474 (the codon encoding amino acid Position 158 (CAG) is marked in grey highlight with bold text). The editable sequence spans nucleotides 475-568; the relevant sequence is shown as the underlined, grey highlight and broken underlined text. In this example, the editable sequence comprises Not1/Hind1 1 1 restriction sites (underlined); an AAVS1 genome editing binding site (grey highlight) and BamH1/Xho1 restriction sites (broken underline). The second reporter sequence is provided by residues 567-814.

As can be seen from the example construct of SEQ ID NO: 5, the editable sequence (nucleotides 475-568) is located between the first and second reporter sequences. As a consequence of the inserted 94bp editable sequence, the second reporter sequence is out of frame with the first reporter sequence.

If one were to translate SEQ ID NO: 5, the resulting sequence would take the form of SEQ ID NO: 6.

SEQ ID NO: 6 - stop codons shown as "dashed lines".

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRY PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADK|AAASLSVPSTPQWGH-GQDW-QKSPILGSLEKERHQGELQDPPQHRGRQRAARRPLPAEHPHRRRPR AAARQPLPEHPVRPEQRPQREARSHGPAGVRDRRRDHSRHGRAVQV

Using general structure 1 outlined above, a construct of this invention may take the following form:

Reporter sequence 1 Reporter sequence 2

GFP N-terminus Editable sequence GFP C-terminus

Residues 1-158 Residues 159-STOP

ATGGTGAGCAAGGGCGAGGAGCTGTTCACC GCGGCCGCAAGCTTATCT AAGAACGGCATCAAGGTGAA GGGGTGGTGCCCATCCTGGTCGAGCTGGAC GTCCCCTCCACCCCACAG CTTCAAGATCCGCCACAACA GGCGACGTAAACGGCCACAAGTTCAGCGTG TGGGGCCACTAGGGACAG TCGAGGACGGCAGCGTGCAG TCCGGCGAGGGCGAGGGCGATGCCACCTAC GATTGGTGACAGAAAAGC CTCGCCGACCACTACCAGCA GGCAAGCTGACCCTGAAGTTCATCTGCACC CCCATCCTTGGATCCCTC GAACACCCCCATCGGCGACG ACCGGCAAGCTGCCCGTGCCCTGGCCCACC GAGA GCCCCGTGCTGCTGCCCGAC CTCGTGACCACCCTGACCTACGGCGTGCAG AACCACTACCTGAGCACCCA TGCTTCAGCCGCTACCCCGACCACATGAAG GTCCGCCCTGAGCAAAGACC CAGCACGACTTCTTCAAGTCCGCCATGCCC CCAACGAGAAGCGCGATCAC GAAGGCTACGTCCAGGAGCGCACCATCTTC ATGGTCCTGCTGGAGTTCGT TTCAAGGACGACGGCAACTACAAGACCCGC GACCGCCGCCGGGATCACTC GCCGAGGTGAAGTTCGAGGGCGACACCCTG TCGGCATGGACGAGCTGTAC GTGAACCGCATCGAGCTGAAGGGCATCGAC AAGTAA TTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAG

SEQ ID NO: 4

SEQ ID NO: 3 In this case, the editable sequence comprises an Adeno-associated virus integration site 1 (AAVS1 ) locus which can be bound by an appropriate TALEN. Once bound, the endonuclease of the TALEN restricts a part of the editable sequence. The resulting double strand break is then repaired by NHEJ which, as stated above, introduces errors and shifts the frame. Again, without wishing to be bound by any particular theory, in the example sequence below (SEQ ID NO: 7) there has been a 1 bp deletion from within the editable sequence (residue 519 ("C") of SEQ ID NO: 5). This restores the GFP N-terminal open reading frame with the C-terminal GFP resulting in the expression of a functional and detectable reporter element.

SEQ ID NO: 7: nucleic acid construct sequence after genome editing event. The sequence comprises a 1 bp deletion at nucleotide position 519; this restores the GFP N- terminal open reading frame with the C-terminal GFP.

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGC CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAAC TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC TACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGGCGGCCGCAAGCTTATCTGTCCCCTCCACCCCACAGTGGGGCCATAGGGAC AGGATTGGTGACAGAAAAGCCCCATCCTTGGATCCCTCGAGAAAGAACGGCATCAAGGTGAACTTCAAGATCCGC CACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTG CTGCTGCCCGACAACCAC TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA SEQ ID NO: 8: Translated version of SEQ ID NO: 7. The amino acid sequence encoded by the inserted (and modified) editable sequence is shown as bold/highlight text.

M V S K G E E L F T G V V P I L V E L D

G D V N G H K F S V S G E G E G D A T Y

G K L T L K F I c T T G K L P V P W P T

L V T T L T Y G V Q c F s R Y P D H M K

Q H D F F K S A M P E G Y V Q E R T I F

F K D D G N Y K T R A E V K F E G D T L

V N R I E L K G I D F K E D G N I L G H

K L E Y N Y N S H N V Y I M A D K Q A A

A S L S V P S T P Q W G H R D R I G D R

K A P s L D P s R K N G I K V N F K I R

H N I E D G s V Q L A D H Y Q Q N T P I

G D G P V L L P D N H Y L s T Q S A L S

K D P N E K R D H M V L L E F V T A A G

I T L G M D E L Y K - In a further aspect, the invention provides a method of assessing or determining the activity or function of a genome editing system, said method comprising:

contacting a genome editing system with a nucleic acid construct according to the first aspect of this invention; and

detecting expression of the encoded reporter element, wherein expression of the encoded reporter element indicates that the genome editing system is functional.

The construct for use in a method according to the second aspect of this invention may be contained within a cell. As such, the method may comprise contacting or introducing a genome editing system with/to a cell containing or harbouring a nucleic acid construct according to the first aspect of this invention.

The cell may be any suitable cell including any suitable mammalian cell. For example, the cell may be a 293FT cell.

A method according to the second aspect of this invention may comprise transfecting a cell with a nucleic acid encoding a genome editing system and a nucleic acid encoding a nucleic acid construct as described in the first aspect of this invention. Nucleic acids encoding genome editing systems and/or constructs according to the first aspect of this invention may be comprised within one or more vector(s), for example one or more expression vector(s). Accordingly, a cell may be transfected with one or more expression vector(s), which vectors encode the construct and genome editing system described herein.

Expression of the reporter element (after a suitable period of incubation (contact) between the genome editing system and the nucleic acid construct) may be detected by any suitable means. For example, reporter element expression may be detected optically and/or with the use of flow cytometry techniques and the like.

In a yet further aspect, this invention provides a kit for assessing or determining the activity or function of a genome editing system, said kit comprising a nucleic acid construct according to the first aspect of this invention. The kit may further comprise a vector into which the nucleic acid encoding the nucleic acid construct may be introduced (cloned). Additionally, or alternatively, the kit may comprise a vector which encodes a nucleic acid construct of this invention. The kit may further comprise buffers and other reagents required in a method according to the second aspect of this invention. The kit may comprise one or more nucleic acid sequences encoding genome editing systems to be tested; such sequences may be provided as isolated sequences and/or together with vectors into which they may be introduced.

DETAILED DESCRIPTION

Figure 1 : GFP-SplitAx - a novel assay for the functional validation of TALENs, zinc fingers and CRISPR. Please note, the term "GFP-SplitAx" is applied to an assay system according to this invention. Moreover, the term "AxTALEN" (see, for example the figures) is applied to specific TALEN molecules generated by the inventors.

Figure 1 a: Schematic of a system according to this invention (designated the "GFP- SplitAx" system). The GFP-SplitAx vector consisting of the N-terminus of GFP (1 -158), a genome editing binding site and the C-terminus (159-end) which is out of frame with the N- terminus. GFP-SplitAx vector with its corresponding genome editing system (for example TALENs AF, AR, OF, OR, zinc fingers, CRISPR) are co-transfected into 293FT cells. The creation of a double strand break and error prone repair by NHEJ can result in deletions or insertions that generate the full length open reading frame of GFP. Figures1 b-1 g: Representative flow cytometry plots of 293FT cells 48 hours after transfection with AAVS1 - GFP-SplitAx only (b), co-transfection of AAVS1 -GFP-SplitAx and AF/AR (c), AAVS1 -GFP- SplitAx and an AAVS ZFN (d), AAVS1 -GFP-SplitAx and AAVS1 CRISPR (e), OCT4-GFP- SplitAx and OF/OR (f) and AAVS1 -Zeis Green-SplitAx and AAVS ZFN (g). Figuresl h -1 1: Graphical representation of flow cytometry data for the GFP-SplitAx and Zeis Green-SplitAx with their respective TALENs, Zinc Fingers or CRISPR. Graphical plots show % GFP or % Zeis Green 293FT cells against cells transfected with a plasmid (+), cells not transfected with a plasmid (-). Data shown as +STDev (n=3).

Figure 2: Targeting of AAVS1 and OCT4 loci in 293FT cells using TALENs. Figure 2a: Schematic overview of the targeting strategy for the AAVS1 locus. The AAVS1 donor plasmid consists of homology arms left (grey box) and right (yellow box) splice acceptor (SA), self-cleaving peptide (2A), puromycin resistance gene (Puro), polyadenylation sequence (PA), pCAG promoter and a fluorescent reporter Zeis Green. Vector specific (A1 , A2) and genomic (A3) PCR primers are indicated. Figure 2b: PCR analysis of genomic DNA isolated from 293FT cells in which the AAVS locus was targeted using the donor plasmid (a) and forward (AF) and reverse (AR) AAVS1 TALENs. Primers pairs designed to amplify a fragment within the donor vector (A1/A2) or from the vector to an external sequence (A1/A3) were used to confirm the correct targeting event. Un-transfected cells (UT), targeting vector only (V), 2 independent experiments with targeting vector and AAVS1 TALENs (V, AF, AR) and negative water control (-ve). Figure 2c: Schematic overview of the targeting strategy for the OCT4 locus. The OCT4 donor plasmid consists of homology arms left (grey box), right (yellow box), exon 5 in frame with the eGFP reporter, Lox P sites (black triangles) encompass a PGK promoter and puromycin resistance gene (Puro). Vector specific primers 01 , 02 and an external genomic primer, 03 are indicated. Figure 2d: PCR analysis of genomic DNA isolated from 293FT 0CT4 targeted cells using the donor plasmid and 0CT4 TALENs OF and OR. Primer pairs designed to amplify within the donor vector (01/02) or from the vector to an external sequence (01/03) were used to the correct targeting event. 293FT cells transfected with single TALEN (OF), Single TALEN (OR), Vector only (V), co- transfection of OCT4 targeting vector, TALEN OCT4-F and -R (OF, OR). Un-transfected cells (UT) and negative water control (-ve).

Figure 3: Schematic of AAVS1 -GFP-SplitAx and validation of SplitAx technology with AAVS1 TALENs, Zinc Fingers and CRISPR. Figure 3a). The GFP-SplitAx vector consisting of the N-terminus GFP (1 -158), AAVS1 binding site and the C-terminus GFP (159-end) which is out of frame with the N-terminus. Co-transfection of the AAVS1 GFP- SplitAx vector with AAVS1 TALEN F and R (rectangle boxes), zinc fingers L and R (dotted lines), CRISPR (T2) (solid line) into 293FT cells. The creation of a double strand break and error prone repair by NHEJ can result in deletions or insertions that restore the GFP open reading frame of the C-terminus with N-terminus. N and X are Not1 and Xho1 restriction sites that allow the binding site to be exchanged for an alternative binding site (See online methods). Figure 3b-l) Flow cytometry of 293FT cells at 48 hours post transfection of the AAVS1 -GFP-SplitAx vector with AAVS1 TALENS, AAVS1 Zinc Fingers and AAVS1 CRISPR. Figure 4: Schematic of OCT4-GFP-SplitAx and validation of the SplitAx technology with OCT4 TALENs. Figure 4a). The GFP-SplitAx vector consisting of the N-terminus GFP (1 -158), OCT4 binding site and the C-terminus GFP (159-end) which is out of frame with the N-terminus. Co-transfection of the OCT4 -GFP-SplitAx vector with OCT4 TALEN F and R (rectangle boxes) into 293FT cells. The creation of a double strand break and error prone repair by NHEJ can result in deletions or insertions that restore the GFP open reading frame of the C-terminus with N-terminus. N and X are Not1 and Xho1 restriction sites that allow the binding site to be exchanged for an alternative binding site. Figure 4b-e) Flow cytometry of 293FT cells at 48 hours post transfection of the OCT4-GFP-SplitAx vector with OCT4 TALENS. Figure 5: AAVS1 -Zeis-SplitAx tested with AAVS1 Zinc Fingers. Flow cytometry of

293FT cells at 48 hours post transfection of the AAVS1 -GFP-SplitAx vector with AAVS1 Zinc Fingers (+) or (-) indicates the whether a plasmid was transfected or not, respectively.

Figure 6: A construct reporter system according to an embodiment of this invention. The construct may be exploited as an assay for the functional validation of genome editing systems. The construct according to this embodiment comprises the N-terminus of GFP (1 - 158), a genome editing sequence (binding site) and the C-terminus (159-end) which is out of frame with the N-terminus. The particular construct has been designated "GFP-SplitAx vector". The GFP-SplitAX vector with its corresponding AxTALENs AF, AR, OF, OR, zinc fingers, CRISPRs are co-transfected into 293FT cells. The creation of a double strand break and error prone repair by NHEJ can result in deletions or insertions that generate the full length open reading frame and restore fluorescence. There are two possible mechanisms the first is that the mutated binding site restores the reading frame with C-terminus of the GFP leading to fluorescent activity. The second mechanism is that the binding site is repaired by NHEJ leading resulting in an ORF but not N-terminal GFP which is in frame with the C-terminal GFP. The N-terminus GFP expressed from a different vector can then complement with the C-terminus GFP of another expressed vector leading to fluorescent activity.

Materials and Methods Nucleic acid construct (Split-Ax) for assessment of genome editing system function

The AAVS1 -GFP-SplitAx and AAVS1 - Zeis GreenSplitAx were generated as a single double stranded DNA oligos (http://eu.idtdna.com/site). 50ng was incubated at 72 °C with dNTP and Taq polymerase (Clontech) to add adenine bases for TA cloning (Life Science). Colonies were grown and plasmid DNA extracted and verified by DNA sequence. The GFP- SplitAx was sub cloned by EcoR1 digest into EcoR1 pCAG-ASIP-ires-Puro. Engineered into this vector immediately upstream and downstream of the genome binding site were Not1 and Xho1 restriction enzyme sites, respectively. These allow the quick exchange of alternative binding sites. The OCT4 genome binding site was cloned into the splitAx vector by Not1 and Xho 1 restriction enzyme cloning.

OCT4-SplitAx design strategy

OCT4 GFP-SplitAx was made by overlapping PCR using Hi Fidelity Taq polymerase (Roche) TA cloned and then sub-cloned by Not1/Xho1 digest into a pCAG-GFP-SplitAx Not1/Xho1 cut vector OCT4-SplitAx_for GCGGCCGCGTCACCTGCAGCTGCCCAGACCTGGC

Notl

Oct4-SplitAx_rev CTCG AG CTG ACCCTG CCTGCTCCTCTCCTG G GTG CCAG GTCTG G G C

Xhol

OCT4-GFP-SplitAx: Amino acid Position 158 marked in bold (see codon "CAG"), followed by Not1 restriction site (under lined). OCT4 genome editing binding site (shaded grey) followed by Xho1 restriction site (broken line). ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGC CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGGCGGCCGCi||Ill||¾|||¾||llIll||l¾||Il^¾II¾¾¾| GCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG CACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC CGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

Plasmid cloning

The pCAG promoter was cloned into the MCS of the plasmid pZDonor-AAVS1 puromycin (Sigma Aldrich) with EcoRV. The Zeis Green-poly A was cloned into the EcoFM site pZDonor-AAVS1 puromycin pCAG. Note the orientation of the pCAG cassette is in the opposite direction to AAVS1 transcription (Figure 2a).

Transfection Protocols

1000ng of TALENs, Zinc Fingers, CRISPR gRNA_AAVS1 -T2 (Addgene Church George), hCAS9 (Addgene Church George) were transfected (Xfect, Clontech) with 500ng respective SplitAx vector into 293FT cells. Flow cytometry was carried out 48 hours post transfection (BD LSR Fortessa) and analysed with FlowJo data analysis Software.

Genome Targeting of the AAVS1/OCT4 loci AAVS1 -TALENs F and R with AAVS1 pZDonor-pCAGASIP-Zeis Green targeting vector and OCT4 TALENs F and R with OCT4-eGFP-PGK-PURO targeting vector(4) (Addgene Jaenisch Rudolph) were introduced into 293 FT cells by Xfect transfection (Clontech). At 72 hours, genomic DNA was isolated (Qiagen DNA extraction kit) for PCR validation assays. Primers used were as follows: For AAVS1 , random insertion A1 -A2 and gene targeted events A1 -A3. For OCT4, random insertion 01 -02 and gene targeted events 01 -03 (see: "list of primers" below).

List of primers

Sequencing primers

M13 F GTAAAACGACGGCCAG

M13 R C AG GAAAC AG C TAT GAC

JDS2978 TTGAGGCGCTGCTGACTG (JOUNG LAB)

JDS2980 T TAAT T CAATATAT TCAT GAGGCAC (JOUNG LAB)

Gbfrag2_for TGGCAATCGCGTCGAACGGGGGAG

Gbfrag3_for GCGATAGCCTCTCATGACGGTGGGA

Gbfragl rev GAGACGCTGAACGGTTTCTAAAGCT

Gbfrag2_rev TGGCAATCGCGTCGAACGGGGGAG

Primers for TALEN preparation

gb1 GACGAGCTGCACCCGCCACTAGCCTATC

gb2 TCATGGCTAACTGCCTTGGTACTGAGC

AAVS1 genomic targeting PCR assay primers

A1 CCGTCGACGCTCTCTAGAGCTAG

A2 TCTCCTGGGCTTGCCAAGGACTCAAAC

A3 CACACCCACACCTGACCCAAACCCAG Oct 4 genomic targeting PCR assay primers

01 CCACTTTGTGGTTCTAAGTACTGTGGTTTC

02 GGCAAGAGAAAGCCTGGTAAACCAGCTAC

03 AACAGGTAACAGCTACATGGTGACT

Results

Functional testing of TALENs with a novel assay (designated: GFP-SplitAx)

Please note, the term GFP-SplitAx is applied to an assay system according to this invention.

To assess the function of the AAVS1 and 0CT4 TALENS, we developed a novel quantitative assay termed GFP-SplitAx. The principle of the assay is that eGFP is split into two fragments consisting of the N-terminus (amino acid 1 -158) and C-terminus (amino acid 159-end) (12,17). These N- and C-terminal fragments are separated by an TALEN binding site such that the C-terminus is out of frame with its N-terminus of GFP. Transfection of the eGFP-SplitAx vector and TALENs introduce double strand breaks which are repaired by error prone non homologous end joining (NHEJ) resulting in deletions or insertions of DNA (8,18). A change in the frame shift in the AAVS1 of -1 , -4 or +1 or +4 or any triplet combination/deletion/insertion of this will restore the open reading frame with the C-terminal eGFP fragment and generate a fluorescent signal within the cell (Figure 1a). Using this biosensor assay, we tested the function of the AAVS1 and OCT 4 TALENs (Figure 3 and Figure 4, respectively). In each case transfection with both forward and reverse TALEN with their respective GFP-SplitAx vectors resulted in GFP reporter activity when compared to control only.

To evaluate whether the GFP-SplitAx could also be used to assess other genome editing tools, we tested AAVS1 Zinc finger nucleases (19) (Figures 1d and 1 i) and an AAVS1 CRISPR (7) (Figures 1e and 1j). Each of these genome editing systems performed well in the GFP-SplitAx assay and the nature of the assay allowed us to quantify their activity (Figure 3). In this assay we demonstrated that the AAVS1 zinc finger nuclease had a higher activity than either CRISPR or TALENs.

To demonstrate that this system could be applied to other loci, we designed a GFP- SplitAx vector containing an OCT4 TALEN binding site.

OCT4-SplitAx design strategy

The OCT4-SplitAx was generated by overlapping primer extension. TA cloned and sub-cloned into a Not1 /Xho1 GFP-SplitAx vector. OCT4-SplitAx_for GCGGCCGCGTCACCTGCAGCTGCCCAGACCTGGC

Not1

Oct4-SplitAx_rev CTCGAGCTGACCCTGCCTGCTCCTCTCCTGGGTGCCAGGTCTGGGC

Xho1

OCT4-GFP-SplitAx: Amino acid Position 158 (CAG) marked in bold/broken underline red, followed by Not1 restriction site (under lined). OCT4 genome editing binding site (shaded grey) followed by Xho1 restriction site (broken line).

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGC CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGGCGGCC^§11||1|1¾11|¾11¾||1|11¾1|¾^¾1|¾¾¾1 GCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG CACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC CGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

Supplementary Figure 11b: Translated 0CT4-GFP-SplitAx:

Amino acid position 158 (grey) and C-terminus out of frame with the N-terminal GFP.

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC M V S K G E E L F T G V V P I L V E L D GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC G D V N G H K F S V S G E G E G D A T Y GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACC

G K L T L K F I C T T G K L P V P W P T CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAG L V T T L T Y G V Q C F S R Y P D H M K CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC Q H D F F K S A M P E G Y V Q E R T I F TTCAAGGACGACGGCAAC TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG F K D D G N Y K T R A E V K F E G D T L GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC V N R I E L K G I D F K E D G N I L G H AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGIIIGCGGCC K L E Y N Y N S H N V Y I M A D K I A A GCGTCACCTGCAGCTGCCCAGACCTGGCACCCAGGAGAGGAGCAGGCAGGGTCAGCTCGA A S P A A A Q T W H P G E E Q A G S A R GAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCA E E R H Q G E L Q D P P Q H R G R Q R A GCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGA A R R P L P A E H P H R R R P R A A A R CAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA Q P L P E H P V R P E Q R P Q R E A R S CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTA H G P A G V R D R R R D H S R H G R A V

CAAGTAA Q V

Translated OCT4-GFP-SplitAx with a 1bp deletion restores GFP N-terminal open reading frame with the C-terminal GFP.

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC M V S K G E E L F T G V V P I L V E L D GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC

G D V N G H K F S V S G E G E G D A T Y GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACC G K L T L K F I C T T G K L P V P W P T CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAG L V T T L T Y G V Q C F S R Y P D H M K CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC

Q H D F F K S A M P E G Y V Q E R T I F TTCAAGGACGACGGCAAC TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG F K D D G N Y K T R A E V K F E G D T L GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC

V N R I E L K G I D F K E D G N I L G H AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGGCGGCC K L E Y N Y N S H N V Y I M A D K Q A A GCGTCACCTGCAGCTGCCCAGACCTGGCCCCAGGAGAGGAGCAGGCAGGGTCAGCTCGAG A S P A A A Q T W P Q E R S R Q G Q L E AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAG

K N G I K V N F K I R H N I E D G S V Q CTCGCCGACCAC TACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC L A D H Y Q Q N T P I G D G P V L L P D AACCAC TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCAC N H Y L S T Q S A L S K D P N E K R D H

ATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC M V L L E F V T A A G I T L G M D E L Y AAGTAA

K -

Co-transfection of OCT4 TALENs (OF and OR) with the OCT4 GFP-SplitAx vector resulted in restoration of GFP fluorescence in a significant proportion of cells that could be monitored by flow cytometry (Figure 4)

The assay (for example the specific "Split-Ax") technology of this invention was found to be applicable to other fluorescent proteins. We designed a Zeis Green Fluorescent protein that was split into two fragments, (N-terminus amino acid 1-158 and the C-terminus amino acid 159-end) separated by the AAVS1 genome editing binding site.

Zeis Green sequence and translated protein atggcccagtccaagcacggcctgaccaaggagatgaccatgaagtac

M A Q S K H G L T K E M T M K Y

cgcatggagggctgcgtggacggccacaagttcgtgatcaccggcgagggcatcggctac

R M E G C V D G H K F V I T G E G I G Y

cccttcaagggcaagcaggccatcaacctgtgcgtggtggagggcggccccttgcccttc

P F K G K Q A I N L C V V E G G P L P F

gccgaggacatcttgtccgccgccttcatgtacggcaaccgcgtgttcaccgagtacccc

A E D I L S A A F M Y G N R V F T E Y P

caggacatcgtcgactacttcaagaactcctgccccgccggctacacctgggaccgctcc

Q D I V D Y F K N S C P A G Y T W D R S

ttcctgttcgaggacggcgccgtgtgcatctgcaacgccgacatcaccgtgagcgtggag

F L F E D G A V C I C N A D I T V S V E

gagaactgcatgtaccacgagtccaagttctacggcgtgaacttccccgccgacggcccc E N C M Y H E S K F Y G V N F P A D G P gtgatgaagaagatgaccgacaactgggagccctcctgcgagaagatcatccccgtgccc

V M K K M T D N W E P S C E K I I P V P

: ji|cagggcatcttgaagggcgacgtgagcatgtacctgctgctgaaggacggtggccgc

I Q G I L K G D V S M Y L L L K D G G R

ttgcgctgccagttcgacaccgtgtacaaggccaagtccgtgccccgcaagatgcccgac

L R C Q F D T V Y K A K S V P R K M P D

tggcacttcatccagcacaagctgacccgcgaggaccgcagcgacgccaagaaccagaag

W H F I Q H K L T R E D R S D A K N Q K

tggcacctgaccgagcacgccatcgcctccggctccgccttgccctga

W H L T E H A I A S G S A L P -

AAVS1- ZeisGreen-SplitAx with AAVSl genome editing binding site downstream of N- terminus (highlighted grey). Stop codons shown as dashes (-). atggcccagtccaagcacggcctgaccaaggagatgaccatgaagtaccgcatggagggc

M A Q S K H G L T K E M T M K Y R M E G

tgcgtggacggccacaagttcgtgatcaccggcgagggcatcggctaccccttcaagggc

C V D G H K F V I T G E G I G Y P F K G

aagcaggccatcaacctgtgcgtggtggagggcggccccttgcccttcgccgaggacatc

K Q A I N L C V V E G G P L P F A E D I

ttgtccgccgccttcatgtacggcaaccgcgtgttcaccgagtacccccaggacatcgtc

L S A A F M Y G N R V F T E Y P Q D I V

gactacttcaagaactcctgccccgccggctacacctgggaccgctccttcctgttcgag

D Y F K N S C P A G Y T W D R S F L F E

gacggcgccgtgtgcatctgcaacgccgacatcaccgtgagcgtggaggagaactgcatg

D G A V C I C N A D I T V S V E E N C M

taccacgagtccaagttctacggcgtgaacttccccgccgacggccccgtgatgaagaag

Y H E S K F Y G V N F P A D G P V M K K

atgaccgacaactgggagccctcctgcgagaagatcatccccgtgcccl|:|i:||gcggccgca

M T D N W E P S C E K I I P V P ! A A A agcttatctgtcccctccaccccacagtggggccactagggacaggattggtgacagaaa

S L S V P S T P Q W G H - G Q D W - Q K

agccccatccttggatccctcgagacagggcatcttgaagggcgacgtgagcatgtacct

S P I L G S L E T G H L E G R R E H V P

gctgctgaaggacggtggccgcttgcgctgccagttcgacaccgtgtacaaggccaagtc

A A E G R W P L A L P V R H R V Q G Q V

cgtgccccgcaagatgcccgactggcacttcatccagcacaagctgacccgcgaggaccg

R A P Q D A R L A L H P A Q A D P R G P

cagcgacgccaagaaccagaagtggcacctgaccgagcacgccatcgcctccggctccgc

Q R R Q E P E V A P D R A R H R L R L R

cttgccctga

L A L

Translated AAVSl-ZeisGreen-SplitAx with a lbp deletion restores Zeis Green N-terminal open reading frame with the C-terminal Zeis Green.

ATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGC

M A Q S K H G L T K E M T M K Y R M E G TGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGC C V D G H K F V I T G E G I G Y P F K G

AAGCAGGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATC

K Q A I N L C V V E G G P L P F A E D I TTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTC L S A A F M Y G N R V F T E Y P Q D I V GACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAG D Y F K N S C P A G Y T W D R S F L F E GACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGAGCGTGGAGGAGAACTGCATG

D G A V C I C N A D I T V S V E E N C M TACCACGAGTCCAAGTTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAG Y H E S K F Y G V N F P A D G P V M K K ATGACCGACAACTGGGAGCCCTCCTGCGAGAAGATCATCCCCGTGCCCAAGGCGGCCGCA

M T D N W E P S C E K I I P V P K A A A AGCTTATCTGTCCCCTCCACCCCACAGTGGGGCCATAGGGACAGGATTGGTGACAGAAAA

S L S V P S T P Q W G H R D R I G D R K GCCCCATCCTTGGATCCCTCGAGACAGGGCATCTTGAAGGGCGACGTGAGCATGTACCTG

A P S L D P S R Q G I L K G D V S M Y L CTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCC

L L K D G G R L R C Q F D T V Y K A K S GTGCCCCGCAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGC V P R K M P D W H F I Q H K L T R E D R AGCGACGCCAAGAACCAGAAGTGGCACCTGACCGAGCACGCCATCGCCTCCGGCTCCGCC

S D A K N Q K W H L T E H A I A S G S A TTGCCCTGA

L P -

AAVS1-Zeis Green-SplitAx synthesised fragment

ATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCAC AAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTGCGTGGTGGAG GGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTAC CCCCAGGACATCGTCGACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAG GACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGTCCAAG TTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCCCTCCTGCGAG

¾||¾¾ ¾^^^¾|¾¾|||GGATCCCTCGAGACAGGGCATCTTGAAGGGCGACGTGAGCATGTACCT GCTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGAT GCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGAACCAGAAGTGGCACCT GACCGAGCACGCCATCGCCTCCGGCTCCGCCTTGCCCTGA

Co-transfection of this vector with the AAVS1 zinc fingers restored Zeis Green fluorescence in a significant proportion of cells (Figure 1g and 1i and Figure 5). Discussion

Split-Ax: novel reporter assay

Technologies including the surveyor mutation detection kit, episomal gene repair assays or homology dependent eGFP repair have been developed to quickly and cheaply screen for the functional activity genome editing systems (14,22-24). Here we describe a novel reporter assay called Split-Ax that can be used to detect the functional activity of Zinc fingers, CRISPR and TALENs. It relies on the basis that a fluorescent protein can be split in to two halves. The N-terminus consists of amino acids 1 -158 whilst the C-terminus is 159- stop codon of eGFP or Zeis Green. The separation of the fluorescent protein N-terminus from the C- terminus by an out of frame genome editing binding site, followed by a double strand break and repair by NHEJ may lead to the C- terminus being in frame with the N- terminus and fluorescence. Although this split eGFP has been used for complementation assays and the split protein phenomenon has also been observed for other fluorescent proteins, this is the first instance to our knowledge where it has been used to assay for genome editing function.

We have shown Spllt-Ax functions with eGFP and Zeis Green. It is likely that this would also be achievable for other fluorescent proteins. It may be possible to use Split-Ax to test for functional activity with different fluorescent proteins to test the efficacy of genome editing tools intended for one step mutations in multiple genes simultaneously (2).

The reporter activity can be monitored in real time, and is both rapid and sensitive.

Detection may be observed at 20 hours visually by microscopy although we routinely assayed by flow cytometry at 48 hours. Accurate quantitative analysis of the genome editing activity can be assessed using flow cytometry. Furthermore, unlike episomal gene repair assays it does not require the generation of two separate vectors namely, a donor vector with homology arms for homology directed repair and the vector containing the binding site. Finally, the genome editing binding site in the Split-Ax vector may be exchanged rapidly through use of the Not1 and Xho1 restriction sites that encompass the genome editing binding site making it an ideal high throughput screening tool.

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Claims

1 . A nucleic acid construct for assessing or determining the expression, activity, function or efficacy of a nucleic acid/genome editing system, said construct comprising a sequence encoding a reporter element and an editable sequence, wherein the editable sequence is contained within the sequence encoding the reporter element.

2. The nucleic acid construct of claim 1 , wherein the reporter element is a fluorescent or luminescent protein.

3. The nucleic acid construct of any preceding claim, wherein the reporter element is selected from the group consisting of:

(i) Green Fluorescent Protein (GFP: or any associated derivatives or variants thereof - for example enhanced (e) GFP);

(ii) Red fluorescent protein;

(iii) Zeis Green Fluorescent protein;

(iv) Lucif erase;

(v) Beta-galactosidase; and

(vi) Beta-glucuronidase.

4. The nucleic acid construct of any preceding claim, wherein the nucleic acid/genome editing system is any system which is capable of manipulating, altering and/or modifying a nucleic acid or genomic sequence.

5. The nucleic acid construct of any preceding claim, wherein the nucleic acid/genome editing system is selected from the group consisting of:

(i) an enzyme or endonuclease;

(ii) a Transcription activator-like effector nuclease (TALEN);

(iii) a recombinase; (iv) zinc finger;

(v) clustered regularly Interspaced short fialindromic repeats (CRISPR); and

(vi) a meganuclease moiety or element.

6. The nucleic acid construct of any preceding claim, wherein the editable sequence comprises a genome editing system binding site and a sequence editable by the genome editing system.

7. The nucleic acid construct of any preceding claim, wherein the sequence encoding the reporter element is split or separated by the editable sequence.

8. The nucleic acid construct of any preceding claim, wherein the editable sequence is flanked by first and second reporter sequences.

9. The nucleic acid construct of claim 8, wherein the first reporter sequence encodes part of a reporter element and the second reporter sequence may encode another part of the same reporter element.

10. The nucleic acid construct of claim 8 or 9, wherein first reporter sequence encodes an N-terminus section of a reporter element and the second reporter sequence encodes a C- terminus section of the same reporter element.

1 1 . The nucleic acid construct of claims 8, 9 or 10, wherein the first and second reporter sequences are out of frame with one another.

12. The nucleic acid construct of any one of claims 8-1 1 , wherein the first reporter sequence encodes the eGFP N-terminus residues 1 -158 and the second reporter sequence encodes the eGFP C-terminus residues 159-END

13. The nucleic acid construct of claim 1 1 , wherein functional expression of the reporter element is achieved by restoring the correct open reading frame between the first and second reporter element sequences.

14. The nucleic acid construct of claim 1 1 , wherein functional expression of the reporter element is achieved through non homologous end joining.

15. The nucleic acid of claim 1 1 , wherein functional expression of the reporter element is not achieved through a single stranded annealing process.

16. The nucleic acid construct of claim 1 1 , wherein the functional expression of the reporter element is not dependent on any donor or companion vector, wherein the donor or companion vector comprises or encodes a copy or part of the reporter element sequence.

17. The nucleic acid construct of any preceding claim, wherein the genome editing system is a TALEN comprising an endonuclease.

18. The nucleic acid construct of claim 17, wherein the endonuclease is a Fok1 endonuclease and the editable sequence comprises a sequence which can be restricted or cut by the Fok1 endonuclease.

19. The nucleic acid construct of any one of claims 8-18, wherein the first reporter sequence comprises, consists or consists essentially of the sequence of SEQ ID NO: 3.

20. The nucleic acid construct of any one of claims 8-19, wherein the second reporter sequence comprises, consists or consists essentially of the sequence of SEQ ID NO: 4.

21 . The nucleic acid construct of any preceding claim, wherein the construct comprises, consists or consists essentially of the sequence of SEQ ID NO: 5.

22. A method of assessing or determining the activity or function of a genome editing system, said method comprising:

contacting a genome editing system with a nucleic acid construct according to any preceding claim; and

detecting expression of the encoded reporter element, wherein expression of the encoded reporter element indicates that the genome editing system is functional.

23. The method of claim 22, wherein the method comprises transfecting a cell with a nucleic acid encoding a genome editing system to be tested and a nucleic acid encoding a nucleic acid construct of any preceding claim.

24. A kit for assessing or determining the activity or function of a genome editing system, said kit comprising a nucleic acid construct according to any preceding claim.

25. Use of the nucleic acid constructs of any preceding claim in a method of assessing or determining whether or not a genome editing system (i) is or has been expressed; and/or (ii) is functional or not.

26. A nucleic acid construct as substantially described herein with reference to the Figures and description.