Patent ID: 11891635
Assignee: MAX-DELBRÜCK-CENTRUM FÜR MOLEKULARE MEDIZIN IN DER HELMHOLTZ-GEMINSCHAFT
Field: Pharmaceuticals (Chemistry)
Classification: CPC C  A | IPC A  C

Claim 0:
1. An in vitro method for modifying a double stranded DNA (dsDNA) molecule in a mammalian cell, the method comprising:
introducing into the cell
i. a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease,
ii. at least two Cas9-specific guide RNAs (guide RNA 1 and 2), and
iii. an exogenous nucleic acid molecule comprising or encoding a DNA substitute sequence,

generating at least two double strand breaks (DSBs) of the dsDNA molecule to be modified, wherein
i. the dsDNA molecule comprises at least two target sequences (target sequences 1 and 2) which are targeted by the at least two Cas9-specific guide RNAs,
ii. wherein guide RNA 1 targets target sequence 1 and guide RNA 2 targets target sequence 2, wherein the guide RNAs are complementary to the corresponding target sequences in the dsDNA molecule,
iii. wherein each target sequence in the dsDNA molecule is located adjacently to a protospacer-adjacent motif (PAM) sequence,
iv. wherein each guide RNA interacts with the Cas9 endonuclease to form a complex that specifically cleaves the dsDNA to create DSBs adjacent to each of the PAM sequences, and

replacing a DNA sequence of the dsDNA molecule to be modified, wherein the replaced sequence is located between the double strand breaks (between target sequences 1 and 2) and wherein the sequence between the DSBs is replaced by the DNA substitute sequence of the exogenous nucleic acid molecule by the non-homologous end joining (NHEJ) pathway, wherein the exogenous nucleic acid molecule comprising or encoding the DNA substitute sequence has nucleotide sequences at its ends that lead to either reconstitution or disruption of the target sequences upon ligation of open ends of the dsDNA molecule to be modified and the DNA substitute sequence, depending on the orientation of the DNA substitute sequence after ligation, wherein if the target sequences are reconstituted after ligation, the DNA substitute sequence can be excised again by the Cas9 endonuclease that is complexed with the Cas9-specific guide RNA and wherein this process can be repeated until the DNA substitute sequence has been inserted in the desired orientation leading to disruption of the target sequences,
i. wherein ligation of the DNA substitute sequence by the NHEJ pathway occurs in orientation 1 or orientation 2,
ii. wherein upon ligation in orientation 1, target sequences 1 and target sequence 2 are restored, and upon ligation in orientation 2, target sequences 1 and target sequence 2 are disrupted.