CREATION AND USE OF GUIDE NUCLEIC ACIDS
Provided herein are methods and compositions to make guide nucleic acids (gNAs), nucleic acids encoding gNAs, collections of gNAs, and nucleic acids encoding for a collection of gNAs from any source nucleic acid. Also provided herein are methods and compositions to use the resulting gNAs, nucleic acids encoding gNAs, collections of gNAs, and nucleic acids encoding for a collection of gNAs in a variety of applications.
1 . A method of making a collection of nucleic acids, comprising:
a. obtaining target nucleic acids, each comprising a PAM site of a nucleic acid-guided nuclease;
b. hybridizing first primers to the PAM sites of the target nucleic acids, wherein the first primers comprise (i) a MAP site that is complementary to the PAM site, (ii) a complementary recognition site that is complementary to a recognition site of the nucleic acid guided nuclease, and (iii) a complementary promoter site that is complementary to a promoter site;
c. extending the first primers using the target nucleic acids as template, thereby producing first extension products comprising sequence of the first primer and sequence complementary to the target nucleic acids;
d. hybridizing second primers to the first extension products; and
e. extending the second primers using the first extension products as template, thereby producing second extension products comprising the PAM site, the recognition site, and the promoter site.
2 . The method of claim 1 , wherein the second primers comprise (i) a PAM site of the nucleic acid-guided nuclease and (ii) a random sequence.
3 . The method of claim 2 , wherein the random sequence is between about 6 and about 8 bases long.
4 . The method of claim 1 , wherein the first primers further comprise a restriction enzyme site of a restriction enzyme.
5 . The method of claim 1 , further comprising:
f. ligating an adapter to the second extension products, wherein the adapter comprises a restriction enzyme site of a restriction enzyme; and
g. cutting the second extension products with the restriction enzyme such that the PAM site and the restriction site are cleaved from the recognition site.
6 . The method of claim 4 or claim 5 , wherein the restriction enzyme comprises MmeI, FokI or MlyI.
7 . The method of claim 1 , further comprising removing unbound first primers or unbound second primers.
8 . The method of claim 1 , wherein the extending the first primers or the extending the second primers is conducted with labeled nucleotides.
9 . (canceled)
10 . The method of claim 1 , wherein the recognition site is about 20 nucleotides in length.
11 . The method of claim 1 , wherein the recognition site is from about 15 to about 25 nucleotides in length.
12 . The method of claim 1 , wherein the nucleic acid-guided nuclease comprises a Cas system protein.
13 . The method of claim 1 , wherein the nucleic acid-guided nuclease comprises a Cas9 system protein.
14 . The method of claim 1 , wherein the target nucleic acids comprise genomic DNA or cDNA.
15 . The method of claim 1 , wherein the target nucleic acids comprise human DNA.
16 - 17 . (canceled)
18 . The method of claim 1 , wherein the complementary recognition site comprises at least one modified nucleic acid bond.
19 . (canceled)
20 . The method of claim 1 , further comprising transcribing the second extension products using the promoter site.
21 . A method of making a collection of nucleic acids, comprising:
a. obtaining target nucleic acids, each comprising a PAM site of a nucleic acid-guided nuclease;
b. hybridizing primers to the PAM sites of the target nucleic acids, wherein the primers comprise (i) a MAP site that is complementary to the PAM site, (ii) a complementary recognition site that is complementary to a recognition site of the nucleic acid guided nuclease, and (iii) a complementary promoter site that is complementary to a promoter site;
c. extending the primers using the target nucleic acids as template, thereby producing extension products comprising the PAM site, the recognition site, and the promoter site;
d. nicking the target nucleic acids; and
e. digesting the nicked target nucleic acids.
22 - 77 . (canceled)
78 . A composition comprising a collection of guide nucleic acids,
wherein each guide nucleic acid comprises a recognition site and a stem loop sequence of a nucleic acid-guided nuclease,
wherein each recognition site is complementary to a target site of a target nucleic acid that is adjacent to a PAM site of the nucleic acid-guided nuclease, and
wherein the target sites to which the recognition sites of the collection of guide nucleic acids are complementary are distributed within the target nucleic acids at an average spacing of less than about 10,000 base pairs.
79 - 254 . (canceled)
255 . The method of claim 1 , wherein:
a) the PAM site comprises NGG or NAG;
b) the collection of nucleic acids comprises at least 10 5 unique nucleic acids; and/or
c) the target nucleic acids are spaced at least every 10,000 bp across a genome of interest.
256 . The method of claim 1 , wherein the target nucleic acids comprise one or more of ribosomal DNA, centromeric DNA, Alu DNA, long interspersed nuclear elements (LINE) DNA, and short interspersed nuc18lear elements (SINE) DNA.