Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Abstract

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeqnuclease digestion and deep sequencing-a massively parallel platform that measures cleavage kinetics and captures the timeresolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a-reported as a more specific nuclease in cells-has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts

“…S2A-C). To enable tracing over the timescale of months, we carefully designed the sgRNAs with nucleotide mismatches to the Target Sites, thus decreasing their affinity (36,37) and slowing the lineage recording rate (22,38).…”

“…S2A-C). To enable tracing over the timescale of months, we carefully designed the sgRNAs with nucleotide mismatches to the Target Sites, thus decreasing their affinity (36,37) and slowing the lineage recording rate (22,38). Approximately 5,000 15 engineered cells ("A549-LT") were then embedded in matrigel and surgically implanted into the left lung of an immunocompromised (C.B-17 SCID) mouse ( Fig.…”

A kinetic model improves off-target predictions and reveals the physical basis ofSpCas9 fidelity

“…Wild type SpCas9 (Cas9 from now on) induces specific double-stranded breaks and the catalytically 'dead' Cas9 (dCas9) mutants allow for binding the target DNA without cleavage 3,5 . Apart from complimentary on-targets, Cas9-sgRNA also binds and cleaves partiallycomplementary off-target DNA sites [11][12][13][14][15][16][17][18] . Off-target cleavage risks unwanted genomic alterations, including point mutations, large-scale deletions, and chromosomal rearrangements 19 .…”

High-throughputin vitrospecificity profiling of natural and high-fidelity CRISPR-Cas9 variants

“…Recent in vitro and in vivo studies profiled kinetic properties and genome editing activities of natural and high-fidelity Cas9 variants (Jones et al, 2019;Schmid-Burgk et al, 2020). In these studies, SpCas9 had high mismatch-tolerance and in turn, more off-target cleavage activity whereas HF Cas9 variants had reduced off-target cleavage activity.…”

“…To avoid these pitfalls, specificity studies can be performed in vitro to detect the native cleavage activities of Cas9 variants (Fu et al, 2019(Fu et al, , 2016(Fu et al, , 2014Huston et al, 2019;Jones et al, 2019;Pattanayak et al, 2013;Zhang et al, 2020). Here, we used a previously established plasmid-based high-throughput in vitro cleavage assay to compare the native cleavage specificity of different Cas9 variants (Murugan et al, 2020).…”