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A recent study by Swedish and British researchers shows that genomic engineering based on CRISPR / Cas9 can cause undesirable effects on the target, and highlights the complexity of the repair mechanisms of human DNA in the presence of the potent prokaryotic nuclease Cas9. The document is currently available on the bioRxiv* preprint server.
Over the past decade, genome engineering efforts have been revolutionized with the increasing use of the CRISPR / Cas9 system. As a result, a myriad of tool sets have been developed, allowing efficient and straightforward loss-of-function disruptions of functional genomic elements.
Therefore, the functionality and applicability of these systems are amazing. In the presence of a guide RNA (gRNA) complementary to the target site and alongside an adjacent protospacer motif (PAM), the expressed Cas9 endonuclease creates a double-stranded break (DSB) at a particular genomic site.
In cases where an exogenous homologous deoxyribonucleic acid (DNA) template is provided, this DSB is restored by homology-directed repair in order to introduce specific mutations or insertions of selected sequences.
However, this is not only valid for deletions of a single nucleotide or of short sequences; longer DNA regions can also be excised from the genome using two gRNAs which flank the targeted region and direct Cas9 to induce two DSBs.
Yet, although a plethora of functional sequences have been deleted from the genome with great success, until now transfer RNA (tRNA) genes have not been targeted in human cells with the use of the. double gRNA system.
These problems were recently addressed by a research group led by Dr Keyi Geng of the Karolinska Institute, Science for Life Laboratory in Sweden, with the ultimate aim of characterizing the genotypic abnormalities induced by Cas9 in human cells.
CRISPR / Cas9 and two-cell systems
In order to study the functionality of the tRNA gene, these researchers deleted two tRNA genes from the genomes of human hyperploid hepatocellular carcinoma (HepG2) and chronic myeloid leukemia haploid (HAP1) cells using the CRISPR / system. Cas9 with two gRNAs.
Specifically, genomic alterations on the target in the aforementioned HepG2 and HAP1 cell clones were analyzed by applying Xdrop-based enrichment for the target region after long read sequencing. In addition, to test the effectiveness of CRISPR / Cas9 in suppressing tRNA genes, the researchers focused on a pair located on human chromosome 17.
The underlying alterations that caused a rearrangement of the region targeted by CRISPR / Cas9 were assessed by a de novo sequence assembly approach. In addition, target insertion events in other confirmed HepG2 and HAP1 deletions were also actively investigated.
Validation of the deletion in HAP1 and HepG2. (A) Schematic illustration of the workflow for obtaining deletion clones derived from single cells. (B) The chromatograms confirm the deletions in the HAP1 Δ72 and HepG2 Δ15 clone. DSB sites induced by CRISPR / Cas9 are represented by red lines and scissors. (CE) Agarose gels confirm the size of the PCR products obtained to validate the deletion events (320 bp) using primers hybridizing to the flanking regions of the target sites (Fig. 1B) in the HAP1 clones (C) and HepG2 (DE). Additional primers to distinguish unmodified control, expected and observed deletion events are used (E). The marker bands specify the size of the DNA in bp. In C, the HAP1 clone with duplicated target regions is in bold. In D, the HepG2 deletion clone is not shown here but in Figure 1C. In E, deletion clones confirmed in D with a reinserted target region (340 bp) are indicated in red.
Assessment of adverse effects on the target
Although a genomic region of interest was cleaved by Cas9 in this study, the researchers showed that the genomic fragment of the target region was not removed from the cell nucleus. Instead, the cleaved fragment was duplicated, inverted, and inserted locally into the genomes of HepG2 and HAP1 cells.
The study also demonstrated the successful integration of exogenous DNA fragments into HepG2 cells. In addition, aberrant DNA fragments derived from targets have been shown to still be functional, labeled with active histones (i.e. proteins that provide structural support for a chromosome) and linked by RNA polymerase. III.
The frequency of targeted genomic alterations and a proposed model. (A) The stacked bar graph indicates the frequency of genomic alterations on the target in the validated HAP1 (n = 5) and HepG2 (n = 17) deletion clones. (BC) Hypothetical model of targeted genomic alterations in HAP1 (B) and HepG2 (C). Cas9 caused the cleavage of DSBs from the target region of the genome. The fragments were inverted and reinserted into a daughter cell (DC). In HepG2, additional exogenous DNA sequences from the genome of E. coli and the Cas9 vector carrying gRNA-1 were integrated into the HepG2 genome downstream of the reinserted fragments derived from the target.
Despite the researchers’ initial attempt to suppress the tRNA genes in order to hinder their expression, aberrant genomic changes found at the original locus resulted in active transcription of the tRNA genes in the two cell types used in this study.
This highlights the fact that CRISPR / Cas9-based genomic engineering can lead to undesirable effects on the target, and that inversion and duplication events (as well as integration of exogenous DNA fragments) can occur in same time.
The consequent specter of the Cas9 suppression system
In conclusion, Xdrop technology in combination with de novo the assembly of long-read sequences surprisingly reveals complex genomic alterations which go hand in hand with CRISPR / Cas9 deletions. These genomic rearrangements do not lend themselves to study only with the use of approaches such as Sanger sequencing, PCR genotyping or standard alignments to the reference genome.
“Our results broaden the spectrum of consequences of the Cas9 suppression system, reinforce the need for meticulous genomic validations, and rationalize additional caution when interpreting the results of a suppression event,” note the study authors in this report. bioRxiv paper.
These results certainly represent a new example of unintended CRISPR / Cas9 editing events that can be overlooked, but which can significantly affect the conclusions drawn from experimental readings. Therefore, these serious concerns should always be taken into account when studying the functionality of modified genomes.
*Important Notice
bioRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information
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