Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas

Abstract

CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicasenuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.

Prophages are associated with extensive CRISPR-Cas auto-immunity

“…Following this first STS, incomplete targeting may lead to further STS expansion by primed spacer acquisition, in type I and II systems (45,46), which will result in the incorporation of multiple new spacers targeting both the prophage and adjacent locations in the bacterial genome. By doing so, the CRISPR system may be preventing prophage induction (47), and perhaps induce prophage clearance or genome deletions (48,49). In many cases however, the result of acquiring STS is an apparent standoff between CRISPR-Cas and targeted prophages, due to mechanisms of auto-immunity avoidance (e.g.…”

Genome editing in mammals using CRISPR type I-D nuclease

“…S5). These features differed from those following mutation by type I-E (Dolan et al 2019), and type II effectors such as Cas9 (Cong et al, 2013;Mali et al, 2013) and Cpf1 (Zetsche et al 2015). Microhomology and insertions were also observed in TiD mutation sites ( Fig.…”

“…We next targeted an internal genomic DNA region corresponding to the TiD gRNA target. We Following a recent genome editing study using CRISPR/Cas type I-E (Dolan et al 2019), we speculated that long-range deletion might be introduced near the TiD target site. To investigate this, we selected targets hEMX1 GTT9(-) for the human EMX1 gene and AAVS GTC_70-107(+) for the AAVS gene based on the luciferase SSA assay ( Fig.…”

“…The CRISPR systems, especially, Class 2 type II systems [CRISPR/Cas9 (Cong et al, 2013;Mali et al, 2013)] and type V [CRISPR/CpfI (Zetsche et al 2015)] have been widely used to efficiently introduce a mutation on a target DNA of interest in eukaryotic chromosomal DNA as a tool of genome editing. Although less common than Class 2 systems, Class 1 type I CRISPR systems have some advantages compared to Cas9 and Cpf1 as genome editing tools, such as a large variety of mutation profiles, including longer gRNA sequences, which might allow higher specificity and longrange genome deletion (Cameron et al 2019;Dolan et al 2019;Morisaka et al 2019).…”

“…Both proteins possess a central helicase motif, and, most importantly, Cas3 also has a nuclease domain, the so-called HD domain. Recent studies have shown that Cas3 is recruited to the R-loop formed by Cascade, and that it nicks non-target strand DNAs, processively degrading DNA in the region upstream of the PAMproximal side (Huo et al 2014;Xiao et al 2018;Dolan et al 2019).…”