Predicting off-target activity of CRISPR-Cas9 complexes


CRISPR-Cas9 is originally a bacterial defense system that uses a guide-RNA to target and nucleases to cut foreign nucleic acids. Recently, these protein-RNA complexes have taken the genome editing world by storm since the revolutionary discovery was made that they could be used as genetic editing tools. By reprogramming the guide-RNA, the Cas9 enzyme could be controlled to cut at a predetermined location along the DNA with high precision. Now known as the molecular scissors of genome editing, the CRISPR-Cas9 enzyme and its engineered variants have opened up the door of endless possibilities: curing genetic diseases, developing cancer therapies, crop enhancements, and much more. However, manipulating CRISPR-Cas9 into human genome editing devices requires thorough characterization of its off-target activities prior to usage. Current methods available are limited in their prediction of off-target DNA cleavage sites and fail to provide any information about biophysical activity. A thorough kinetic analysis of how these protein complexes bind and cleave at off-target DNA sequences remains elusive.


Researchers at the University of Texas at Austin have developed a method known as NucleaSeq1: a rapid, massively parallel in vitro platform that measures cleavage kinetics of CRISPR-Cas nucleases. This method is able to capture time-resolved identities of the Cas9 nuclease cleavage products for over 10,000 targets containing mismatches, insertions, and deletions relative to the guide RNA. In order to comprehensively evaluate five engineered and natural Cas9 variants for DNAs containing guide-RNA-relative mismatches, insertions, and deletions, the researchers coupled NucleaSeq with their other technology, CHAMP2. CHAMP enabled the researchers to comprehensively profile the Cas9 binding patterns from an Illumina chip and compare this data to the NucleaSeq results. Most notably, merging these high-throughput data sets of cleavage specificity (NucleaSeq) and binding specificity (CHAMP) for each of the five Cas9 variants, allowed the researchers to create biophysical model that compares each of these nucleases directly, revealing mechanistic insights into off target cleavage. More broadly, NucleaSeq and CHAMP enable rapid, quantitative, and systematic comparisons of the specificies and cleavage products of CRISPR nucleases. As CRISPR-Cas9 systems continue to be developed for human gene modification, NucleaSeq and CHAMP are paving the way for rapid and quantitative determination of off-target binding sites in patient-specific genomes.


1.  Jones, S.K., Hawkins, J.A., Johnson, N.V. et al. Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nat Biotechnol 39, 84–93 (2021).

2.  Jung C, Hawkins JA, Jones SK Jr, Xiao Y, Rybarski JR, Dillard KE, Hussmann J, Saifuddin FA, Savran CA, Ellington AD, Ke A, Press WH, Finkelstein IJ. Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips. Cell. 2017 Jun 29;170(1):35-47.e13.