COMPOSITIONS AND METHODS FOR THE SELF-INACTIVATION OF BASE EDITORS
The invention features polynucleotides that encode bases editors having a heterologous intron for self-inactivation, compositions comprising such polynucleotides, and methods of inactivating a base editor encoded by such polynucleotides.
1 . A polynucleotide encoding a deaminase domain, a nucleic acid programmable DNA binding protein (napDNAbp) domain, or a base editor polypeptide or fragment of any of the foregoing, the polynucleotide comprising an intron, wherein the intron is inserted in an open reading frame encoding the deaminase, napDNAbp, the base editor polypeptide, or fragment thereof.
2 . The polynucleotide of claim 1 , wherein the intron comprises an alteration at a splice acceptor or splice donor site, wherein the alteration reduces or eliminates splicing of base editor mRNA, thereby reducing or eliminating expression of a base editor polypeptide.
3 . The polynucleotide of claim 1 , wherein a base editor comprises the nucleic acid programmable DNA binding protein (napDNAbp) domain or deaminase domain.
4 . A polynucleotide encoding a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain or a deaminase domain, the polynucleotide comprising an intron, wherein the intron is inserted in an open reading frame encoding the napDNAbp domain or the deaminase domain.
5 . The polynucleotide of claim 7 , wherein the intron comprises an alteration at a splice acceptor or splice donor site that reduces splicing of the base editor mRNA.
6 . The polynucleotide of claim 5 , wherein the deaminase domain is a cytidine deaminase domain or an adenosine deaminase domain.
7 . The polynucleotide of claim 1 , wherein the intron is derived from a sequence selected from the group consisting of NF1, PAX2, EEF1A1, HBB, IGHG1, SLC50A1, ABCB11, BRSK2, PLXNB3, TMPRSS6, IL32, ANTXRL, PKHD1L1, PADI1, KRT6C, and HMCN2.
8 . The polynucleotide of claim 1 , wherein the intron comprises a sequence that has at least about 85% nucleic acid sequence identity to one of the following:
a)
(SEQ ID NO: 226)
GTGAGATCAAATGAAAGTTTCATATAGAAATACAAAACCTAGAGAACTGGCATGT
AAGAGAAGCAAAAATTACTTCAGCAAGGCCATGTTAGTAAATTTGCATCTGTTTGTCCACAT
TAG;
b)
(SEQ ID NO: 227)
GTAGGTGACAATGCTGCAGCTGCCTAATCTAGGTGGGGGGAACTAAATTGTGGGT
GAGCTGCTGAATGGTCTGTAGTCTGAGGCTGGGGTGGGGGGAGACACAACGTCCCCTCCCTG
CAAACCACTGCTATTCTGTCCCTCTCTCTCCTTAG;
c)
(SEQ ID NO: 228)
GTAAGTGGCTTTCAAGACCATTGTTAAAAAGCTCTGGGAATGGCGATTTCATGCT
TACATAAATTGGCATGCTTGTGTTTCAG;
d)
(SEQ ID NO: 229)
GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGT
CTAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTT
GCCTTTCTCTCCACAG;
e)
(SEQ ID NO: 230)
GTAAGCACAACTGGGATGGGGTGACAGGGGTGCAAGATTGAAAACTGGCTCCTCT
CCTCATAGCAGTTCTTGTGATTTCAG;
f)
(SEQ ID NO: 231)
GTAAGAAATGTTATTTTTCAGTAAGTGATTTAGTTATTTTTCCTTTTTTCTCATTA
AAATTTCTCTAACATCTCCCTCTTCATGTTTTAG;
g)
(SEQ ID NO: 232)
GTGAGACCCTAGCCCCCTCAACCCTGCCCTGGCCTCTCCCCAAACCTGCCCCCCC
ACGCTGACCCCCACACCCGGCCGCCCGCAG;
h)
(SEQ ID NO: 233)
GTGGGTGTCAGAGGCATCGGGGCTGCGGGGTAGGGGGCTGCCCCACCCCTAACGA
AGTCTGCTCCTCCAG;
i)
(SEQ ID NO: 234)
GCAGGGAAGTCCTGCTTCCGTGCCCCACCGGTGCTCAGCTGAGGCTCCCTTGAAAA
TGCGAGGCTGTTTCCAACTTTGGTCTGTTTCCCTGGCAG;
j)
(SEQ ID NO: 235)
GTGGGGAGTTGGGGTCCCCGAAGGTGAGGACCCTCTGGGGATGAGGGTGCTTCTCT
GAGACACTTTCTTTTCCTCACACCTGTTCCTCGCCAGCAG;
k)
(SEQ ID NO: 236)
GTATAGACCCCTTGATCTCCTAACCCTAACCCTAACCCTAACCCTAACCTACAAA
ATCTTAGAGCATCAGTGGGAGCATCTCACTGTCCAGGCTCAATATTTCTTCATTTTCTTGCA
G;
l)
(SEQ ID NO: 237)
GTAATTATGATTAAAGATGGTGATTGTTTATTTTCTTTTATGATTGTCCTTAGTAT
TATGTAACCTGCAAATTCTATTGCAG;
m)
(SEQ ID NO: 238)
GTGAGTGACACAAGGTGTTGTCTGGGGAGTGGGGAAGGGGGATGGAAGTGAATCC
TGTTGGTGGGGTGGAGAAAGGGCGATCTCAAGAGGGCCACTCTCTCCAG;
n)
(SEQ ID NO: 239)
GTAAGCATCTCCACCATCCTTCTGTTTACTCTGATGGGGTCTGCAAAGGGGAGAT
GATGTATAGGGTTGGGTATCTCTGTAAATGTCAGATGTGAAGTTGATCTTATGACCTTCTGT
TCTGCAG;
o)
(SEQ ID NO: 240)
GTGAGGGTCTCCCAGGCTGGGCAGGGGGAGGGGGCTGCTGCCTTGATTGCGTCCC
AGGACACAGCCCTCCTCCAGCCTGCCCTCGCCTTGCTCATCCCCTCCCCATCTCAGCCCCAC
CCCCACTAACTCTCTCTCTGCTCTGACTCAG;
p)
(SEQ ID NO: 241)
GTAATGATTGATTGCAATGTATGATTACAATAATCTCAGTATAAGTTCAGTAATA
ATAACCTTCCACTGCTGTCCTCTGTGTGCACCCAG;
or
q)
(SEQ ID NO: 242)
GTAAATATATACAACAGTTTTTCATTTAAATAAGTGCACGGCACAAATAAGAAAA
ATATGTCAAAAATGTAACCAATAGTTTTTTTCAAATTTAG.
9 . A composition or pharmaceutical composition comprising:
(i) a first polynucleotide encoding a deaminase domain and an N-terminal fragment of a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the N-terminal fragment of the napDNAbp domain is fused to a split intein-N, and
(ii) a second polynucleotide encoding a C-terminal fragment of the napDNAbp domain, wherein the C-terminal fragment of the napDNAbp domain is fused to a split intein-C;
wherein the first or second polynucleotide comprises an intron, wherein the intron is inserted in an open reading frame of the polynucleotides.
10 . A base editor system comprising:
(i) a polynucleotide encoding a base editor comprising an adenosine deaminase domain, or fragment thereof, comprising an altered catalytic residue;
(ii) one or more guide RNAs that direct the base editor to edit a site in the genome of a cell; and
(iii) one or more guide RNAs that direct the base editor to edit the polynucleotide encoding the base editor, wherein the edit results in a decrease in activity and/or expression of the encoded base editor; or
(i) a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof;
(ii) one or more guide RNAs that direct the self-inactivating base editor to edit a site in the genome of a cell; and
(iii) one or more guide RNAs that direct the self-inactivating base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide encoding the self-inactivating base editor.
11 . The base editor system of claim 10 , wherein the altered catalytic residue of the deaminase domain is His57 (H57), Glu59 (E59), Cys87 (C87), or Cys90 (C90) of the following reference sequence:
(SEQ ID NO: 1)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI
GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSR
IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCY
FFRMPRQVFNAQKKAQSSTD,
or a corresponding position in another adenosine deaminase.
12 . A base editor system comprising:
(i) a first polynucleotide encoding a deaminase domain and an N-terminal fragment of a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the N-terminal fragment of the napDNAbp domain is fused to a split intein-N;
(ii) a second polynucleotide encoding a C-terminal fragment of the napDNAbp domain, wherein the C-terminal fragment of the napDNAbp domain is fused to a split intein-C,
wherein the first or second polynucleotide comprises an intron, wherein the intron is inserted in an open reading frame, and wherein the first and second polynucleotides encode a base editor;
(iii) one or more guide RNAs that direct the base editor to edit a site in the genome of a cell; and
(iv) one or more guide RNAs that direct the base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (i) or (ii); or
(i) a first polynucleotide encoding an N-terminal fragment of a deaminase domain, wherein the N-terminal fragment of the deaminase domain is fused to a split intein-N;
(ii) a second polynucleotide encoding a C-terminal fragment of the deaminase domain and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the C-terminal fragment of the deaminase domain is fused to a split intein-C,
wherein the first or second polynucleotide comprises an intron, wherein the intron is inserted in an open reading frame, and wherein the first and second polynucleotides encode a base editor;
(iii) one or more guide RNAs that direct the base editor to edit a site in the genome of a cell; and
(iv) one or more guide RNAs that direct the base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (i) or (ii).
13 . The base editor system of claim 12 , wherein the base editor system comprises a polynucleotide sequence selected from the following:
a)
(SEQ ID NO: 191)
gGUUUUAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
b)
(SEQ ID NO: 192)
gUUUCUUACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
c)
(SEQ ID NO: 193)
gGUUUCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
d)
(SEQ ID NO: 194)
GCCACUUACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
e)
(SEQ ID NO: 195)
gACAUUAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
f)
(SEQ ID NO: 196)
gGAUCUCACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
g)
(SEQ ID NO: 197)
gUCCUUAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
h)
(SEQ ID NO: 198)
GUCACCUACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
i)
(SEQ ID NO: 190)
GAUUUCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
j)
(SEQ ID NO: 200)
gGUGCUUACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
k)
(SEQ ID NO: 201)
gUCCACAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
l)
(SEQ ID NO: 202)
GAUACUUACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
m)
(SEQ ID NO: 203)
gUGUUUUAGCUGCGGCAAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
n)
(SEQ ID NO: 204)
gUUUCUUACAGCCAUAAUUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
p)
(SEQ ID NO: 205)
gCUCCACAGCUGCGGCAAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
p)
(SEQ ID NO: 206)
GAUACUUACAGCCAUAAUUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
q)
(SEQ ID NO: 207)
gUGUUUUAGGGACGAAAGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
r)
(SEQ ID NO: 208)
gUUACCUGGCUCUCUUAGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
s)
(SEQ ID NO: 209)
gCUCCACAGGGACGAAAGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
t)
(SEQ ID NO: 210)
gCUUGCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
u)
(SEQ ID NO: 211)
gAUUGCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
v)
(SEQ ID NO: 212)
gUCUCCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
w)
(SEQ ID NO: 213)
gUCUGCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
x)
(SEQ ID NO: 214)
gGACUCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
y)
(SEQ ID NO: 215)
GCACCCAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
z)
(SEQ ID NO: 216)
gAAUUUAGGUCAUGUGUGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
aa)
(SEQ ID NO: 217)
gCAUUAGGUCGAGAUCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
bb)
(SEQ ID NO: 218)
gCCUUAGGUCGAGAUCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
cc)
(SEQ ID NO: 219)
GUUUCAGGUCGAGAUCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
dd)
(SEQ ID NO: 220)
gACAUUAGGCUAAGAGAGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ee)
(SEQ ID NO: 221)
gUCCUUAGGCUAAGAGAGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ff)
(SEQ ID NO: 222)
gGUUUCAGGCUAAGAGAGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
gg)
(SEQ ID NO: 223)
gACAUUAGAUUAUGGCUCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
hh)
(SEQ ID NO: 224)
gUCCUUAGAUUAUGGCUCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ii)
(SEQ ID NO: 225)
gGUUUCAGAUUAUGGCUCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
jj)
(SEQ ID NO: 524)
gCACCAUGAGCGAGGUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
kk)
(SEQ ID NO: 525)
gGCCACCAUGAGCGAGGUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ll)
(SEQ ID NO: 526)
GUGUCGAAGUUCGCCCUGGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
mm)
(SEQ ID NO: 527)
gAUGCCGAGAUAAUGGCCCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
nn)
(SEQ ID NO: 528)
gAUGCCGAGAUAAUGGCCCUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
oo)
(SEQ ID NO: 529)
gAUGCCGAGAUCAUGGCACUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
pp)
(SEQ ID NO: 530)
gAUGCCGAGAUCAUGGCACUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
qq)
(SEQ ID NO: 531)
gAUGCCGAGAUCAUGGCACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
rr)
(SEQ ID NO: 532)
gAUGCCGAGAUCAUGGCGCUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ss)
(SEQ ID NO: 533)
gAUGCCGAGAUCAUGGCGCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
tt)
(SEQ ID NO: 534)
gAUGCCGAGAUCAUGGCGUUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
uu)
(SEQ ID NO: 535)
gAUGCCGAGAUUAUGGCACUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
vv)
(SEQ ID NO: 536)
gAUGCCGAGAUUAUGGCACUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ww)
(SEQ ID NO: 537)
gAUGCCGAGAUUAUGGCACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
xx)
(SEQ ID NO: 538)
gAUGCCGAGAUUAUGGCACUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
yy)
(SEQ ID NO: 539)
gAUGCCGAGAUUAUGGCGCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
zz)
(SEQ ID NO: 540)
gAUGCCGAGAUUAUGGCUCUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
aaa)
(SEQ ID NO: 541)
gAUGCGGAGAUCAUGGCGCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
bbb)
(SEQ ID NO: 542)
gAUGCUGAGAUAAUGGCCCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ccc)
(SEQ ID NO: 543)
gAACCGCACAUGCCGAAAUUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
ddd)
(SEQ ID NO: 544)
gGCAGGUGUCGACAUAUCUAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
eee)
(SEQ ID NO: 545)
gAUGCCGAAAUUAUGGCUCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
fff)
(SEQ ID NO: 546)
gACACAUGACACAGGGCUCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG
CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU;
or
ggg)
(SEQ ID NO: 547)
gGCCCCAGCACACAUGACACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG
GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU.
14 . A vector comprising a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof.
15 . A cell comprising a vector comprising a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof.
16 . A kit comprising the polynucleotide of claim 1 .
17 . A method for reducing or eliminating expression of a self-inactivating base editor, the method comprising:
(a) providing a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof; and
(b) contacting the polynucleotide with a guide RNA and a self-inactivating base editor polypeptide, wherein the guide RNA directs the base editor to edit a splice acceptor or a splice donor site of the intron, thereby generating an alteration that reduces or eliminates expression of the self-inactivating base editor.
18 . A method of self-inactivating base editing, the method comprising:
(a) expressing in a cell a polynucleotide encoding a base editor comprising a deaminase domain, or fragment thereof;
(b) contacting the cell with a first guide RNA that directs the base editor to edit a site in the genome of the cell, thereby generating an alteration in the genome of the cell; and
(c) contacting the cell with a second guide RNA that directs the base editor to edit the polynucleotide encoding the base editor, wherein the edit results in a decrease in activity and/or expression of the encoded base editor, thereby generating an alteration that reduces or eliminates expression of the base editor; or
(a) expressing in a cell a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof;
(b) contacting the cell with a first guide RNA that directs the self-inactivating base editor to edit a site in the genome of the cell, thereby generating an alteration in the genome of the cell; and
(c) contacting the cell with a second guide RNA that directs the self-inactivating base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (a), thereby generating an alteration that reduces or eliminates expression of the self-inactivating base editor.
19 . A method of editing the genome of an organism, the method comprising:
(a) expressing in a cell of the organism a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof;
(b) contacting the cell with a first guide RNA that directs the self-inactivating base editor to edit a site in the genome of the cell, thereby generating an alteration in the genome of the cell; and
(c) contacting the cell with a second guide RNA that directs the self-inactivating base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (a), thereby generating an alteration that reduces or eliminates expression of the self-inactivating base editor; or
(a) expressing in a cell of the organism a first polynucleotide encoding a deaminase domain and an N-terminal fragment of a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the N-terminal fragment of the napDNAbp domain is fused to a split intein-N, and a second polynucleotide encoding a C-terminal fragment of the napDNAbp domain, wherein the C-terminal fragment of the napDNAbp domain is fused to a split intein-C, wherein the first or second polynucleotide comprises an intron, wherein the intron is inserted in an open reading frame, and wherein expression of the first and second polynucleotides in the cell result in the formation of a self-inactivating base editor;
(b) contacting the cell with a first guide RNA that directs the self-inactivating base editor to edit a site in the genome of the cell, thereby generating an alteration in the genome of the cell; and
(c) contacting the cell with a second guide RNA that directs the self-inactivating base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (a), thereby generating an alteration that reduces or eliminates expression of the self-inactivating base editor; or
(a) expressing in a cell of the organism a first polynucleotide encoding an N-terminal fragment of a deaminase domain, wherein the N-terminal fragment of the deaminase domain is fused to a split intein-N, and a second polynucleotide encoding a C-terminal fragment of the deaminase domain and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the C-terminal fragment of the deaminase domain is fused to a split intein-C, wherein the first or second polynucleotide comprises an intron, wherein the intron is inserted in an open reading frame, and wherein expression of the first and second polynucleotides in the cell result in the formation of a self-inactivating base editor;
(b) contacting the cell with a first guide RNA that directs the self-inactivating base editor to edit a site in the genome of the cell, thereby generating an alteration in the genome of the cell; and
(c) contacting the cell with a second guide RNA that directs the self-inactivating base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (a), thereby generating an alteration that reduces or eliminates expression of the self-inactivating base editor.
20 . A method of treating a subject, the method comprising:
(a) expressing in a cell of the subject a polynucleotide encoding a self-inactivating base editor or fragment thereof, wherein the polynucleotide comprises an intron inserted in an open reading frame of the self-inactivating base editor or fragment thereof;
(b) contacting the cell with a first guide RNA that directs the self-inactivating base editor to edit a site in the genome of the cell, thereby generating an alteration in the genome of the cell to treat the subject; and
(c) contacting the cell with a second guide RNA that directs the self-inactivating base editor to edit a splice acceptor or a splice donor site present in the intron of the polynucleotide of (a), thereby generating an alteration that reduces or eliminates expression of the self-inactivating base editor.
21 . The method of claim 20 , wherein the intron comprises a sequence that has at least about 85%, 90%, 95%, or 99% nucleic acid sequence identity to one of the following:
a)
(SEQ ID NO: 226)
GTGAGATCAAATGAAAGTTTCATATAGAAATACAAAACCTAGAGAACTGGCATGT
AAGAGAAGCAAAAATTACTTCAGCAAGGCCATGTTAGTAAATTTGCATCTGTTTGTCCACAT
TAG;
b)
(SEQ ID NO: 227)
GTAGGTGACAATGCTGCAGCTGCCTAATCTAGGTGGGGGGAACTAAATTGTGGGT
GAGCTGCTGAATGGTCTGTAGTCTGAGGCTGGGGTGGGGGGAGACACAACGTCCCCTCCCTG
CAAACCACTGCTATTCTGTCCCTCTCTCTCCTTAG;
c)
(SEQ ID NO: 228)
GTAAGTGGCTTTCAAGACCATTGTTAAAAAGCTCTGGGAATGGCGATTTCATGCT
TACATAAATTGGCATGCTTGTGTTTCAG;
d)
(SEQ ID NO: 229)
GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGT
CTAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTT
GCCTTTCTCTCCACAG;
e)
(SEQ ID NO: 230)
GTAAGCACAACTGGGATGGGGTGACAGGGGTGCAAGATTGAAAACTGGCTCCTCT
CCTCATAGCAGTTCTTGTGATTTCAG;
f)
(SEQ ID NO: 231)
GTAAGAAATGTTATTTTTCAGTAAGTGATTTAGTTATTTTTCCTTTTTTCTCATTA
AAATTTCTCTAACATCTCCCTCTTCATGTTTTAG;
g)
(SEQ ID NO: 232)
GTGAGACCCTAGCCCCCTCAACCCTGCCCTGGCCTCTCCCCAAACCTGCCCCCCC
ACGCTGACCCCCACACCCGGCCGCCCGCAG;
h)
(SEQ ID NO: 233)
GTGGGTGTCAGAGGCATCGGGGCTGCGGGGTAGGGGGCTGCCCCACCCCTAACGA
AGTCTGCTCCTCCAG;
i)
(SEQ ID NO: 234)
GCAGGGAAGTCCTGCTTCCGTGCCCCACCGGTGCTCAGCTGAGGCTCCCTTGAAAA
TGCGAGGCTGTTTCCAACTTTGGTCTGTTTCCCTGGCAG;
j)
(SEQ ID NO: 235)
GTGGGGAGTTGGGGTCCCCGAAGGTGAGGACCCTCTGGGGATGAGGGTGCTTCTCT
GAGACACTTTCTTTTCCTCACACCTGTTCCTCGCCAGCAG;
k)
(SEQ ID NO: 236)
GTATAGACCCCTTGATCTCCTAACCCTAACCCTAACCCTAACCCTAACCTACAAA
ATCTTAGAGCATCAGTGGGAGCATCTCACTGTCCAGGCTCAATATTTCTTCATTTTCTTGCA
G;
l)
(SEQ ID NO: 237)
GTAATTATGATTAAAGATGGTGATTGTTTATTTTCTTTTATGATTGTCCTTAGTAT
TATGTAACCTGCAAATTCTATTGCAG;
m)
(SEQ ID NO: 238)
GTGAGTGACACAAGGTGTTGTCTGGGGAGTGGGGAAGGGGGATGGAAGTGAATCC
TGTTGGTGGGGTGGAGAAAGGGCGATCTCAAGAGGGCCACTCTCTCCAG;
n)
(SEQ ID NO: 239)
GTAAGCATCTCCACCATCCTTCTGTTTACTCTGATGGGGTCTGCAAAGGGGAGAT
GATGTATAGGGTTGGGTATCTCTGTAAATGTCAGATGTGAAGTTGATCTTATGACCTTCTGT
TCTGCAG;
o)
(SEQ ID NO: 240)
GTGAGGGTCTCCCAGGCTGGGCAGGGGGAGGGGGCTGCTGCCTTGATTGCGTCCC
AGGACACAGCCCTCCTCCAGCCTGCCCTCGCCTTGCTCATCCCCTCCCCATCTCAGCCCCAC
CCCCACTAACTCTCTCTCTGCTCTGACTCAG;
p)
(SEQ ID NO: 241)
GTAATGATTGATTGCAATGTATGATTACAATAATCTCAGTATAAGTTCAGTAATA
ATAACCTTCCACTGCTGTCCTCTGTGTGCACCCAG;
or
q)
(SEQ ID NO: 242)
GTAAATATATACAACAGTTTTTCATTTAAATAAGTGCACGGCACAAATAAGAAAA
ATATGTCAAAAATGTAACCAATAGTTTTTTTCAAATTTAG.