Highly efficient therapeutic gene editing of human hematopoietic stem cells
Authors: Qiuming Yao
Kendell Clement
David M Dorfman
Alessandra Biffi
Luca Pinello
Daniel E Bauer
Luca Biasco
Benjamin P Roscoe
John P Manis
Mitchel A Cole
Pengpeng Liu
Carlo Brugnara
Jing Zeng
Anne H Shen
Christian Brendel
Cristina Baricordi
Cicera R Lazzarotto
David A Williams
Scot A Wolfe
Chunyan Ren
Kevin Luk
Shengdar Q Tsai
Erica B Esrick
Yuxuan Wu
Journal: Nature Medicine (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: γ-globin genes; HBG1/2; β-globin disorders; sickle cell disease; β-thalassemia
Affiliations: Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA., Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA., Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China., Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA., Department of Pathology, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA., Department of Pathology, Brigham and Women's Hospital , Boston, MA, USA., Department of Pathology, Harvard Medical School, Boston, MA, USA., University College of London, Great Ormond Street Institute of Child Health, Faculty of Population Health Sciences, London, UK., Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA. ., Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. ., Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA. .

Abstract:  Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction. 

Retracted: No

PubPeer comments:
 Translocations abound in this human hematopoietic stem cells CRISPR study I have reported that the vector in integrating within the genome in almost every study. Here I could identify beta-lactamases, but not along with the BCL11a gene. One can argue that it is sequencing artifacts. So, here are translocations. In fact, looking at those I can tell where the gRNAs have been designed to cut. An example of a translocation from BCL11A (BCL11A), RefSeqGene on chromosome 2 with chromosome 9 SRR8509791.82836.2 82836 length=150 CAGAAAAGAGATATGGCATCTACTATAAAAGCAACTGTTAGCTTGCACTAGACTAGCTTCAAAGTA TATTTGGATGTTCCAGAAGCCCACAAGCACCTCACATTCAAATAGCTGAAACAGCCTATCACCTTTC CTCAGACCTGAATCTCC ![file] 25 (ATAAAAGCAACTGTTAGCTTGCACTAGACTAGCTTCAAAGT) 65 matches 100% to the BCL11A gene in chromosome 2 66 (ATATTTGGATGTTCCAGAAGCCCACAAGCACCTCACATTCAAATAGCTGAAACAGCCTAT CACCTTTCCTCAGACCTGAATCTCC) 150 matches 100% to chromosome 9 Note, that the cut is exactly where the sgRNAs are defined. Here is list of them based on the NG_011968.1 Homo sapiens BAF chromatin remodeling complex subunit BCL11A (BCL11A), RefSeqGene on chromosome 2 63160 ACTCTTAGACATAACACACC 63179 63161 CTCTTAGACATAACACACCAG 63181 63222 GTTGCTTTTATCACAGGCTCC 63242 ....... 63311 ATCGGTGGCCGTTTGCCCAGG 63331 LIST of 47 other sequences with translocations SEQSAB3253 AAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACC SEQSAB10052 AACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCT SEQSAB14448 AAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGG SEQSAB14489 AAGCAAATAACAATAATAATAAATAAAATAATTTGGGTTAAGTGTAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGGGGGGCCTCTTTCGGAAGGCT SEQSAB17871 AATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCA SEQSAB18290 AATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGG SEQSAB65627 ACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTC SEQSAB104002 AGAGCCTTCCGAAAGAGGCTCCCCCGTGCAAACGGCCACCGATGGAGAGTGCTGCCAGTCCTCTTCTACCCCACCCACGCCCCCACCCTAATGCCCCTGCAGCATCTCTGTTCAGGTGACCGAGCATGGCCTGATTCCCTGCCACTGTAG SEQSAB120350 AGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTT SEQSAB120560 AGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGT SEQSAB136164 AGCTTTCTGGTTAGATTGTTTTCACTTGGTGATCATGTCTTTTCCATGTGTACCTGTAATATTTTTCCATCATTCACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCT SEQSAB216592 CAAGCAATTCTCCTGCCTCAGCCTCCTGAGTAGCTGAGATTACAGGCACCTGCCACAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGGGGGGCCTC SEQSAB263846 CACCCTGGAAAACAGCCTGACTCCCCTCTCCCAAAGCAGAAATTCTCTTCGTGACGCAAGCTAACAGTTGCAACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCA SEQSAB279616 CAGAATCATTTGTCACTCACAGTTCAACCTAGACTTACCTGGTAATTGGAGTGGCCATCTGAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCGGACCTCTCCATCGGTGGCCGTTTGCCCAGG SEQSAB316685 CATTCCTTAATAGGCTCCGGAAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGG SEQSAB317490 CCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCC SEQSAB318964 CCACGCCCCCACCCTAATCAGAGGCCAAACCCTTCCTGGAGCCGAGTTAGCAGGGAGAAGGAGGCTCTCAGTGGGGCGGGGGTTGAGGGTGAGGGAAGGCTTCACAATGAAGGTGCCATTTGAATGGGTCTTAAGGATGGGAAGGATTTG SEQSAB321188 CCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCTACCTGCCTTGGCCTCCCAAAGTGCTGGGATTATCACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATC SEQSAB321666 CCAGTCCCTCTATCTGGCCTCTATCTATAAGGACTGTGGTCAGTTGTCACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGGGGGG SEQSAB325699 CCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCT SEQSAB337693 CCTGGTGCAGATCAGCATGGTTTGGTACAATCTGGTGCAGATCAGAGTGATTTGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGGGG SEQSAB392177 CTTAATAGGCTCCGGAAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAA SEQSAB410478 GACAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAACAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATAACCAGGACACAACCAAGCCACAGAGATTCTGTGGAACCTGACTGCAGGGGTCA SEQSAB424203 GAGAGGTCTGCCAGTCCTCTTCTACCCCACCCACGCCCCCACCCTAATCAGAGGCCAAACCCTTCCTGGAGCCTGGGCCCACGGGTTTGGGGCCTTATTGTTCGCCGTTGCTGTTCTGGCTGACCCACTGGTGCTGAGGCCTGGCTGGCA SEQSAB494957 GCATGAGAACTGGGACCTGGGTCCAGCCCAGTCCATGGTGGCCACAGCTGGATCTCAGCTCTGCTCCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGT SEQSAB509521 GCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCC SEQSAB510133 GCCACTGCTCAGAAAGGAAGTACAAGATGCTCCTGTTCTTATTGATGGAGTAAAAAGAGGCTGATATGATGGCTGTCTTACTCACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGC SEQSAB516260 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAACAGGGTAAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATTGACATGCTGCCAGTGCAAGCCAGGCCTCAGCACCAGTGGGTCAGCCAGAACAG SEQSAB516809 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAACAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATAAACCTACAGCTCCAAAATCATTCTTTTTAAATCCTTGTCCCACATCTAGGACA SEQSAB516956 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAACAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATGAGGCGGGCGGATCATCAAGATCCTCCTGGCCAACATGGTGAAACCACGTCTCT SEQSAB517089 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAACAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATTGACATCCTGCAAGTGCCAGCCAGGCCTCAGCACCAGTGGGTCAGCCAGAACAG SEQSAB517334 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAGCAGGGTCAATACAACTTAGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATAACTAGAGCTGTCCAACAACCAAGGAGATAGCTGGATGTGAAAAGTCTGCCATC SEQSAB517372 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAGCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATAATGTAGAAGGATGTCAGGGACAGTCATTTTTGACCTGAAATAGCAACCTCATG SEQSAB517546 GCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACATCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATATTTACGCTCCTACCAGCAATGCATGAGGGCACCAATTCCCCACACCCCCACCA SEQSAB522212 GCCAGAAAAGAGATATGGCATCTACTCTTAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTTGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCT SEQSAB523584 GCCAGAAAAGAGATATGGCATTGGGCCACAAGCCAAGGAATGAGGATAACCTCTAGAAACTGGAAAAGATAGGAAACTGATTAGGGTGGGGGCGTAGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGGG SEQSAB707670 TAAAGATTACAGATTCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATC SEQSAB712424 TACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGCCGTTTGCCCAGGGG SEQSAB714418 TAGGCTCCTGAAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGA SEQSAB727811 TCCAGAAAAGAGATATGGCATCTACTCTTAGACATAACACAACAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATAGTAAAACCTAAATCCTTGGAGTGGCCTTCAAGTTCCTACACTTTCTGGCCCCT SEQSAB739767 TCCCAGCTTCTCGGGAGGCTGAGGCAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGCAAGCCGAGACCACGCCACTGCACTTCAGCCTGGGCGCGAGACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGG SEQSAB740640 TCCGGAAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGC SEQSAB741614 TCCTTAATAGGCTCCGGAAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAG SEQSAB812724 TGCCCTTGCTCATTCCTTAATAGGCTCCGGAAACCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCG SEQSAB817209 TGTACTGTGCAAGGTATTTGCTATAAGAAACCCAGAACTGTCAAATTATAAACTCGCAAGGGACTCTGGTGCCTAGTTAAGTTTCCCACCCTAATCAGAGGCCAACCCTTCCTGGAGCTAAAAGCAACTGTTAGCTTGCACTAGACTAGC SEQSAB827560 TTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTAGAAGAGGACTGGCAGACCTCTCCATCGGTGGC SEQSAB832117 TTCCTTAATAGGCTCCGGAAACCCAATAAAGATTACAGATGCCAAATACGAGACTAGACTCCAAATGAACTGTTTTGTAAATGTATCCCAACTAACACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGTGGGGGCGTGGGTGGGGTA , 
  Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility, and cancers. And you can see[ from this paper] which starts with the above,  chromosomal translocations in embryonic stem cells by CRISPR Cas9 in mice. And yet a [sickle-cell patient is being made to go through this ] - without proper information and consent. , 
 Not to say that translocations don t happen, but these are very unlikely to be translocations. This data was generated by amplicon sequencing, which would not amplify translocations. Thus, these are most likely insertions. Also, it is very unlikely that ex vivo gene therapy of hematopoeitic stem cells will lead to infertility and congenital disorders due some cells with translocations. Cancer is a risk, and it is likely monitored in these clinical trials. I agree that we should be prudent with gene editing because there s a lot of unknowns. Nonetheless, if you read the article you posted, you d notice that SCD patients are ready to go through a lot to have a chance to be cured (or at least reduce complications), because of the toll the disease has on their lives.  without proper information and consent . How do you know? , 
 I say that because [20 21 ppl died] in PD-1 CRISPR Chineses trial - and [hardly any press] (or [citations] ). If you knew, I apologize for that  assumption and take back my words. Whether insertions or translocations, point is that both genes have been cut - an indirect proof of [off-targets] . We dont know whats happening in the [rest of the genome] . , 
 [Amplicon-seq] is the best way to analyze off-targets, better than [nanopore] . The data here shows so many off-targets - here is one with [8 mismatches, 18 reads] in two samples. With 8 mismatches one essentially has possible 100000 off-targets. Can our cells take this? ![file] , 
 The chromosomes are literally getting shredded. These are the genes that integrate with BCL11a (so only 2% of the genome is being looked at). And also this is amplicon-seq - ie we are only looking at BCLL1a. Any of the other two genes can also integrate. The number of reads is given just before the accid 1. 225 NM_032134.2 glutamine rich 2 (QRICH2), 2. 165 NM_001286462.1 chromosome 21 open reading frame 58 (C21orf58), 3. 111 NR_046235.3 RNA, 45S pre-ribosomal N5 (RNA45SN5), ribosomal RNA 4. 98 NM_194449.4 PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), mRNA 5. 58 XM_011544683.1 PREDICTED: fibroblast growth factor 17 (FGF17), 6. 45 NM_015124.5 GRAM domain containing 4 (GRAMD4), 7. 42 XM_024452143.1 PREDICTED: basic proline-rich protein-like (LOC112268284), mRNA 8. 32 NM_006887.5 ZFP36 ring finger protein like 2 (ZFP36L2), mRNA 9. 15 NM_152240.2 zinc finger matrin-type 3 (ZMAT3), 10. 12 NM_001170544.1 PGAM family member 5, mitochondrial serine threonine protein phosphatase (PGAM5), 11. 9 NM_003661.3 apolipoprotein L1 (APOL1), 12. 8 NM_172177.4 mitochondrial ribosomal protein L42 (MRPL42), 13. 6 NM_001079880.1 protein kinase D2 (PRKD2), 14. 5 NM_033540.3 mitofusin 1 (MFN1), mRNA 15. 5 NM_144597.3 chromosome 15 open reading frame 40 (C15orf40), 16. 5 NM_001164281.2 TRAF3 interacting protein 2 (TRAF3IP2), 17. 4 NM_005154.5 ubiquitin specific peptidase 8 (USP8), 18. 2 NM_001142643.2 CASK interacting protein 2 (CASKIN2), 19. 2 NM_001193609.1 tubulin delta 1 (TUBD1), 20. 2 NM_001322415.1 PNN interacting serine and arginine rich protein (PNISR), , 
 And if you use plasmid (unlike here, which used protein electroporation) it will [integrate the plasmid] - at different points - showing these must have been edited too. This difference is interesting - showing the cell [picks up whatever is close to patch up the broken chromosome] . Plasmid is more easily available, if not there it will pick up other edited chromosomes. , 
 From Kiran Musunuru twitter thread: [ This is unambiguously an off-target mutation. JK dismisses it out-of-hand: it was far away from any known gene, u201cso we concluded that this intergenic off-target genomic insertion might not impact any biological functionu201d. If JK thinks it isnu2019t impactful, it doesnu2019t count? ] Just see how many off-targets are there, two posts up. And off-targets are so hard to [observe even with deep WGS] - and I am sure He didnt spend much time on it. , 
 And [chromosomal translocations] in the 3 patients in the UPenn study is just a side-note - you have seen the quanta of damage (2 posts up)? Can our cells, esp the immune ones, take that? Feel sad - if any of these scientists had half a brain and 1 10 th of a heart, they would have known the [harm they are doing] . , 
 And this [chromosome shredding] is what the venerated [George Church calls  genome vandalism ] , 
 This chromosome shredding leads to:  [we show] that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs ,