Tuesday, July 8, 2014

Low rate of #CRISPR off-target mutations in human iPS cells, reported in 2 new papers.

Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Talkowski ME, Musunuru K.   Low Incidence of Off-Target Mutations in IndividualCRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected by Whole-GenomeSequencing.  Cell Stem Cell. 2014 Jul 3;15(1):27-30.

Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang K, Cheng L, Ye Z.   Whole-Genome Sequencing Analysis Reveals HighSpecificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs.  Cell Stem Cell. 2014 Jul 3;15(1):12-3.

These papers are very important for using WGS to thoroughly catalog all variants in iPS cells post-CRISPR (and TALENs).  Good news:  Very very low rate of off-target (OT) CRISPR mutations, in contrast to some previous reports of high OT rates in transfected cells.   The authors suggest that the discrepancy may exist because the other studies used different, more commonly-used, "workhorse", non-stem, transformed/quick replicating cell lines.  It's possible that there are some technical differences in transfections and/or specific CRISPR reagents that contribute to these differences, but it is nice to see two different groups in agreement on the iPS situation.  Also, this is reminiscent of the observation by several groups that CRISPR OT effects in mice (generated by zygote injection of CRISPR reagents) are also very minimal.

So this is the not-so-good news, not for CRISPR per se, but for clonal propagation of iPS cells in general:  Both groups discovered that iPS clones accumulated numerous non-CRISPR-related new mutations.  That is, the act of isolating and passaging clonal cell lines itself led to accumulation of 50-100 new single-nucleotide variants not seen in the parental cell line.  This is genome-wide, so only a few of these are likely to be within exons, but still.     The bottom line is that iPS subclones are not, strictly speaking, genetically identical to the parent cell or each other.  Whether this is going to be a major problem going forward in the iPS field remains to be seen.

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