Genome-wide detection of CRISPR editing in vivo using GUIDE-tag

Abstract

Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.

Optimization of NLS Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells

“…We assayed for active off-target sites for 3xNLS enAspCas12a and SpyCas9 with corresponding guide RNAs by GUIDE-tag genome-wide specificity analysis in HEK293T cells. 37 A small number of potential off-target sites were identified for both SpyTS1 and enAspTS2 target sites as determined by Unique Molecular Identifier (UMI) count in GUIDE-tag analysis (Fig. 4B; Supplementary Table S6 [SpyTS1]; Supplementary Table S7 [EnAspTS2]).…”

“…The GUIDE-tag raw sequencing data pre-processing and analysis pipeline is available at https://github.com/locusliu/GUIDESeq-Preprocess_from_Demultiplexing_to_Analysis and as we previously reported. 37,39,40 Briefly, it consists of the following steps: Demultiplexing and UMI extraction. Raw BCL files were converted and demultiplexed using the appropriate i5 and i7 sequencing barcodes, allowing up to one mismatch in each barcode.…”

“…37 Libraries were constructed as previously described. 37 Briefly, 200 ng of genomic DNA was incubated with 2 μL of assembled transposome at 55 °C for 7 min, and the product was cleaned up (20 μL) with a Zymo column (Zymo Research, #D4013). Tagmented DNA was used for the 1 st PCR using PlatinumTM SuperFi DNA polymerase (Thermo) with i5 primer and GUIDE-Tag specific primers (Supplementary Table S5).…”

“…The GUIDE-tag raw sequencing data pre-processing and analysis pipeline is available at https://github.com/locusliu/GUIDESeq-Preprocess_from_Demultiplexing_to_Analysis and as we previously reported. 37,39,40 Briefly, it consists of the following steps: I.…”

“…Adaptor oligonucleotides were synthesized by IDT (Supplementary Table S4). 37,38 Transposon assembly was done by incubating 158 μg Tn5 with 1.4 nmol annealed oligo (contains the fulllength Illumina forward (i5) adapter, a sample barcode, and unique molecule identifier (UMI) at room temperature for 60 min. 37 Libraries were constructed as previously described.…”