Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq

Abstract

Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.

Figure 1.. Schematic workflow of GUIDE-seq method.

The GUIDE-seq procedure is divided into 5 sections with the protocol step numbers as shown on the left: 1) In vitro transcription (IVT) and ribonucleoprotein (RNP) complexation, 2) nucleofection, cell culturing and genomic DNA (gDNA) isolation; 3) NdeI restriction and indel analysis, 4) GUIDE-seq library preparation, and 5) next-generation sequencing and data analysis. Original figure.

Figure 2.. Principle of GUIDE-seq library preparation and paired-end analysis.

(i) Genomic DNA is sonicated to average size of 500 bp, followed by a standard library generation steps (end repair, A-tailing and adapter ligation); (ii) PCR1 with primer complementary to the ligated adapter on one side and either sense D or antisense R primer complementary to dsODN sequence on the other. The opposite strands dsODN primers are used in separate PCR reactions; (iii) Nested PCR2 includes an additional primer containing p7 index and complementary to the dsODN primer on its 3-prime. i5 index encoded in amplified part of the adapter. Each sample has unique combination of i5 and i7 indices, allowing for parallel sequence and analysis of numerous samples; (iv) library normalization is required for balanced representation of samples and opposite strands for each sample; (v) Library sequencing and analysis, the reads are grouped by unique combination of p5 (blue) and p7 (purple) indices, indexes are marked yellow, dsODN sequence is red. Bidirectionally mapped indicate DSB locations. Original figure.

Figure 3.. Quality control steps in the GUIDE-seq protocol.

(a) NdeI analysis of dsODN incorporation on Agilent TapeStation. Profile of uncut PCR amplicon (blue) is superimposed with profile of the same PCR product digested with NdeI (red). Positions of the molecular size marker are indicated at the bottom. The targeted PCR is encompassing B2M site 6 as described by Lazzarotto et al.. (b) Agilent TapeStation analysis of DNA shearing distribution that shows a peak with median size centered around ~450 bp. Original figure.

Figure 4.. GUIDE-seq example output.

GUIDE-seq on-target and off-target detailed visualizations are shown from previously published experiments (a) low-specificity gRNA targeting AAVS1 site 4, (b) high-specificity gRNA targeting PTPN6 site 2. Each row represents an on-target or off-target site and GUIDE-seq read counts corresponding to that site are shown to the right. A dot represents matches with respect to the intended target sequence, and colored nucleotides indicate the sequence of mismatches. Visualizations are ordered by GUIDE-seq read counts. Manhattan plots for (c) AAVS1 site 4 and (d) PTPN6 corresponding to the same GUIDE-seq data are shown; x-axis represents chromosomal location and y-axis represents GUIDE-seq read counts. Original figure.