CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering

Figure 5

Strategies for improving the CRISPR/Cas9 specificity. (a) A pair of sgRNAs guiding Cas9 nickases to bind and nick the opposite DNA strands complementary to gRNA sequences. (b) Fusion of catalytically “dead” Cas9 with dimerization-dependent FokI nuclease domains. (c) Altering the gRNA to truncated gRNA (truRNA) with only17-18 nucleotides. (d) gRNA with two additional guanine nucleotides at the 5′- end to form 5′-GGX20 sgRNA. Engineered variants of Cas9: (e) SPCas9-HF1, (f) eSpCas9, and (g) HypaCas9 (in silico mutants were generated from RCSB PDB-4008 using Discovery Studio).

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