Programmable endonucleases have increasingly become an important tool for targeted genome engineering or modification in eukaryotes. Recently, RNA-guided clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) systems have emerged as a new generation of genome modification tools. These new programmable endonucleases have greatly improved the genome editing capability compared to previous generations of nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).
However, not all genomic targets are accessible to efficient modification by these programmable endonucleases. In fact, some CRISPR-Cas endonucleases appear to have little or no activity in human cells. Among other things, chromatin structure may present a barrier to these programmable endonucleases and prevent them from binding the target sequence. Thus, there is a need for improving accessibility of these programmable endonucleases to target sequences and/or improving the efficiency of targeted genome modification. Moreover, there is a need for increasing the specificity to targeted genome modification by reducing off-target effects.