Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Abstract

Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

Multiplex genotyping method to validate the multiallelic genome editing outcomes using machine learning-assisted long-read sequencing

“…As long-read sequencing induces base calling errors across a segment and cannot be used as is to validate the genome editing outcomes (22), novel screening techniques and tools need to be developed in order to identify diverse sequence changes in the genome. Short-range PCR amplification and Sanger sequencing confirmed that no additional mutation was detected in 'Intact' alleles identified by DAJIN, suggesting that DAJIN validates the consequences of genome editing at the base level (Figs.…”

“…Furthermore, there is a possibility of gene conversion between homologous regions following genomic DNA cleavage [11][12][13]. Although targeted long-read sequencing allows the detection of complex ontarget mutations over several kilobases [9,14], this method has instrumental limitations such as error rates that need to be overcome to validate the target locus to a singlebase level [15].…”

“…Thus, the DAJIN workflow is considerably shorter than that of conventional methods.DNA double-strand break repair leads to long-range deletion, inversion, and insertion as well as small indels in zygotes and stem cells[8,9,[35][36][37]. As long-read sequencing induces base-calling errors across a segment and cannot be used as is to validate the genome editing outcomes[15], novel screening techniques and tools need to be developed in order to detect diverse sequence changes in the genome. A parallel analysis of short-and long-read sequencing results confirmed that DAJIN could identify editing outcomes, including unpredictable large-scale inversion events, in mouse zygotes (Additional file 1:Figs.…”

The phosphoinositide 5-phosphatase INPP5K: From gene structure to in vivo functions

“…We have generated Inpp5k floxed and then Inpp5k Δ/Δ mice and we could confirm that the latter mice died during embryonic life, as Pps Brdm1/Brdm1 mice. Our floxed mice as well as those recently generated by McCabe (McCabe et al, 2019) will certainly help to answer to most of the above questions and will probably allow discovering the exact INPP5K function during eye development and in lens cortex, neurons and brain as well as skeletal muscle. The analysis of INPP5K floxed mice will probably reveal a wider phenotype than in INPP5K patients, with alterations involving more biological processes and organs, given that human INPP5K mutations do not completely abolish the 5-phosphatase catalytic activity or function.…”

Anticipating and Identifying Collateral Damage in Genome Editing

“…Crucially, no single technology is able to capture all of the unexpected sequence changes that can result from genome editing. Targeted sequencing using Sanger, or next-generation methods [26,58,71], affords validation of the targeted locus to the single-base level, but only reports on sequence variation at loci that are chosen as relevant and on the integrity of an interval of a limited size; neither do these techniques identify additional sequence changes elsewhere. Droplet digital PCR [27,39] or even Southern blot analysis [41] help to identify unexpected copy numbers of given sequences, but do not report on the exact sequences.…”