Multiplexed analysis of neuron projections by sequencing
The present invention provides a composition comprising a plurality of labeled neurons, each of which is labeled by an expression construct that encodes a unique barcoded nucleic acid.
1. A composition comprising a plurality of labeled neurons, each of which contains
(a) a barcoded nucleic acid which serves as a label; and
(b) a chimeric protein containing
(i) a nucleic acid binding domain that is specifically bound to the barcoded nucleic acid in the labeled neuron; and
(ii) a synaptic trafficking signal sequence capable of directing transport of the barcoded nucleic acid to the axon terminals of the labeled neuron, wherein at least 50% of the plurality of labeled neurons are labeled with a unique barcoded nucleic acid, and wherein the barcoded nucleic acid contains a binding region that is specifically recognized by the nucleic acid-binding domain of the chimeric protein.
2. The composition of claim 1 , wherein the binding region is a boxB motif or an MS-2 stem-loop motif.
3. The composition of claim 1 , wherein the chimeric protein is a modified presynaptic protein.
4. The composition of claim 1 , wherein the presynaptic protein is pre-mGRASP.
5. The composition of claim 1 , wherein the nucleic acid-binding domain is an nλ domain or a portion of the MS-2 coat protein.
6. The composition of claim 1 , wherein the chimeric protein further comprises a Myc epitope tag and/or a CLIP-tag domain.
7. The composition of claim 1 , wherein the chimeric protein is MAPP-nλ or CLAP-1x-nλ.
8. The composition of claim 1 , wherein the barcoded nucleic acid and chimeric protein are encoded by a modified Sindbus virus expression construct.
9. The composition of claim 8 , wherein the modified Sindbus virus is produced using a defective helper RNA which produces virions that are and neurotropic propagation-incompetent.
10. The composition of claim 9 , wherein the modified Sindbus virus is produced using the defective helper RNA DH-BB (5′ SIN;TE12ORF) .
11. The composition of claim 1 , wherein the barcode in each of the barcoded nucleic acids has a length of (k) nucleotides, wherein 4 k is greater than the number of neurons to be labeled.
12. The composition of claim 1 , wherein the barcoded nucleic acid contains a barcode region that is about 30 nucleotides in length.
13. The composition of claim 1 , wherein the barcoded nucleic acid encodes a fluorescent marker.
14. The composition of claim 1 , wherein the barcoded nucleic acid is RNA.
15. A method for obtaining a plurality of labeled neurons according to claim 1 , comprising infecting neurons with a modified, barcoded Sindbus virus library.
16. The composition of claim 1 , wherein at least 75% of the plurality of labeled neurons are labeled with a unique barcoded nucleic acid.
17. The composition of claim 1 , wherein at least 95% of the plurality of labeled neurons are labeled with a unique barcoded nucleic acid.