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in some embodiments, a stretch of no more than 10, 9, 8, 7, 6 contiguous T bases) may be substituted with a non-T nucleotide, e.g., an A, so that the string is broken down into smaller stretches of Ts with each stretch having 4, or fewer than 4 (for example, 3 or 2) contiguous Ts

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In particular embodiments, 16 guide RNA nucleotides at the 3 end are replaced with DNA nucleotides

(a) Schematic of bicistronic expression vector (PX330) for U6 promoter-driven single guide RNA (sgRNA) and CBh promoter-driven human codon-optimized Figure 20A-G Streptococcus pyogenes Cas9 ( hSpCas9 ) used for all subsequent experiments