High-throughput profiling of mismatched sgRNAs

As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in a variety of biotechnology applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, greatly limits its usage. Besides, understanding the quantitative effects of mismatch can guide the rational design of sgRNAs with tailored activity. In this work, We generated two sgRNA libraries carrying saturated single- and double-nucleotide mismatches. Based on a massively parallel approach, we profiled the in vivo binding affinity of dCas9 towards the DNA target guided by each individual sgRNA with particular mismatch mutations.