A method of generating an optimized guide RNA (gRNA), the method comprising: a) identifying a target region of interest, the target region of interest comprising a protospacer sequence; b) determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; c) determining at least one or more off-target sites for the full-length gRNA; d) generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer targeting sequence or segment, the RNA segment is at the 5' end or the 3' end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 5' end or the 3' end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; e) calculating an estimate of or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the invasion kinetics and lifetime are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; f) comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; g) randomizing 1 to N nucleotides in the linker and 1 to M nucleotides in the RNA segment of the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; h) identifying an optimized gRNA, wherein the optimized gRNA has a binding lifetime greater than or equal to that of the full-length gRNA at the protospacer, and/or a binding lifetime less than or equal to that of the full-length gRNA at the off-target site; and i) testing the optimized gRNA in vitro to determine the specificity of binding. a) identifying a target region of interest, the target region of interest comprising a protospacer sequence; b) determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; c) determining at least one or more off-target sites for the full-length gRNA; d) generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer targeting sequence or segment, the RNA segment is at the 5' end or the 3' end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 5' end or the 3' end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; e) calculating an estimate of or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the invasion kinetics and lifetime are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; f) comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; g) randomizing 1 to N nucleotides in the linker and 1 to M nucleotides in the RNA segment of the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; h) identifying an optimized gRNA, wherein the optimized gRNA has a binding lifetime greater than or equal to that of the full-length gRNA at the protospacer, and/or a binding lifetime less than or equal to that of the full-length gRNA at the off-target site; and i) testing the optimized gRNA in vitro to determine the specificity of binding.