US 20180291382A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0291382 A1 Bier et al. (43 ) Pub . Date : Oct. 11, 2018 ( 54 ) METHODS FOR AUTOCATALYTIC Publication Classification GENOME EDITING AND NEUTRALIZING (51 ) Int. Ci. AUTOCATALYTIC GENOME EDITING C12N 15 / 63 (2006 . 01 ) C12N 15 / 10 ( 2006 .01 ) (71 ) Applicant : The Regents of the University of C12N 9 / 22 ( 2006 .01 ) California , Oakland , CA (US ) CI2N 15 / 11 (2006 . 01 ) C12N 15 /82 ( 2006 .01 ) ( 52 ) U . S . CI. (72 ) Inventors : Ethan Bier , San Diego , CA (US ) ; CPC . .. C12N 15 /63 (2013 . 01) ; C12N 15 / 102 Valentino Gantz , La Jolla , CA (US ) ( 2013. 01 ) ; C12N 2310 /20 ( 2017 . 05 ) ; C12N 15 / 11 ( 2013 .01 ) ; C12N 15 /8279 ( 2013 .01 ) ; ( 21 ) Appl. No .: 15 /524 , 440 C12N 9 /22 ( 2013 .01 ) (22 ) PCT Filed : Nov . 4 , 2015 (57 ) ABSTRACT The invention provides methods for autocatalytic genome (86 ) PCT No. : PCT/ US15 /58961 editing based on genomic integration of a construct contain § 371 ( C ) ( 1 ) , ing multiple elements . More specifically , the invention pro ( 2 ) Date : May 4 , 2017 vides a method for autocatalytic genome editing or for deleting or neutralizing autocatalytic genome editing based on the CRISPR / Cas9 system , and methods of use thereof, in animals, humans , and plants for eliminating pathogens , Related U . S . Application Data targeting suppression of crop pests , strategies to combat (60 ) Provisional application No. 62 / 075 ,534 , filed on Nov . virus ( e . g . , HIV ) and other diseases ( e . g . , cancer ) caused by 5 , 2014 , provisional application No . 62 / 101, 443 , filed retrovirus, as well as to generate , and reverse , homozygous on Jan . 9 , 2015 . mutations that are transmitted to nearly all offspring . Patent Application Publication Oct . 11 , 2018 Sheet 1 of 8 US 2018 / 0291382 A1 te ! HWA rzitt! FIGURE 1A FIGURE 1B ** * 59 2 . * * * * * * * * * * * * ** * * ** * * * * * 1929 FIGURE 1C FIGURE 1D FIGURE 1E FIGURE 1F ?????????????????????????????????? PA FIGURE 1G Patent Application Publication Oct. 11 , 2018 Sheet 2 of 8 US 2018 /0291382 A1 VINA WWW 2222222 FIGURE 2A FIGURE 2B UH . FIGURE 2C LLLLLLLLLLLL riiiiiiiiiiiiii ILUL FIGURE 2D Patent Application Publication Oct . 11 , 2018 Sheet 3 of 8 US 2018 / 0291382 A1 FIGURE 2E . : : . FIGURE 2F FIGURE 2G FIGURE 2H WWW WW : : : : : : . : FIGURE 21 Patent Application Publication Oct . 11 , 2018 Sheet 4 of 8 US 2018 / 0291382 A1 12: . }}** * * * ww . Wilmin31 . : : . ' : . : . ' . ' , ' 1 ' , ' . FIGURE 3A FIGURE 3B . : ... : : . ' . ' . ' . rec *** * ** * * - * FIGURE 3C .: :. : . :'. :. ili 22 LUB . :. .i' ::: : FIGURE 3D Patent Application Publication Oct . 11 , 2018 Sheet 5 of 8 US 2018 / 0291382 A1 .1 . Writ :2 FIGURE 4A FIGURE 4B WWWWWWW . .. : : . : . :. : . : . : : FIGURE 4C FIGURE 4D XXX FIGURE 4E FIGURE 4F . FIGURE 4G Patent Application Publication Oct. 11 , 2018 Sheet 6 of 8 US 2018 / 0291382 A1 Traditional Mendelian Genetics Active Genetics . ?? FIGURE 5 ???? ????????? MA cerent 2 FIGURE 6 Patent Application Publication Oct . 11 , 2018 Sheet 7 of 8 US 2018 / 0291382 A1 ?? FIGURE 7A ???? ?????? ? ???? ?? ? ? ??? ????? ????????????????? ?????? ? ?? . : ?? : :??? ????? : ???:?? ????????????? ???. ???? ??? + + + + + ?? : : : * * FIGURE 7B Patent Application Publication Oct . 11 , 2018 Sheet 8 of 8 US 2018 / 0291382 A1 Example. : . : . : . of CHACR element MORQX Wt; knoc blishini .?????????????????????????????? WWWY7vvvvvvv *** ** ?????? ???????????????? * * * Ni * FIGURE 8 ????????????????? ? :???????????????????????????????????????????????????????????????????????????? WWW ::: : ::: : !; FIGURE 9 US 2018 /0291382 A1 Oct. 11 , 2018 METHODS FOR AUTOCATALYTIC Elements for Reversing the Autocatalytic Chain Reaction GENOME EDITING AND NEUTRALIZING (ERACRs ) , can be comprised of a number of elements AUTOCATALYTIC GENOME EDITING whereby to inject the construct as a DNA plasmid together with a plasmid source of Cas9 protein into the germline of CROSS -REFERENCE TO RELATED an organism and obtain transgenic organisms carrying this APPLICATIONS insertion . Organisms carrying this construct would then be [0001 ] This application claims the priority benefit of U . S . crossed to MCR individuals ( or released into an environ provisional application Ser . Nos . 62 /075 ,534 and 62 / 101 , ment containing MCR individuals ) whereupon NCR would 443 , filed Nov . 5 , 2014 and Jan . 9 , 2015 , respectively . The act on the MCR chromosome to delete the MCR element and entire contents of which are hereby incorporated by refer could also restore function of the host locus via a recoded ence herein . transgene . [0007 ] The present inventions are based on a well -known STATEMENT AS TO FEDERALLY SPONSORED bacterial immunity function known as the CRISPR /Cas9 RESEARCH system that is based on two components . The first compo nent is an endonuclease such as Cas9 , that has a binding site [0002 ] This invention was made with government support for the second component , which is the guide polynucleotide under Grant Nos . A1070654 and NS029870 awarded by the ( e . g ., guide RNA ) . The guide polynucleotide ( e . g . , guide National Institutes of Health . The government has certain RNA ) directs the endonuclease ( e . g . , Cas9 ) protein to DNA rights in the invention . templates ( e . g . , a bacteriophage integrated into the bacterial chromosome) based on sequence homology. The Cas9 pro BACKGROUND OF THE INVENTION tein then cleaves that template leading to secondary muta [ 0003] CRISPRs (clustered regularly interspaced short tions during DNA repair. The CRISPR /Cas system has been palindromic repeats ) are DNA loci containing short repeti used for gene editing ( e . g . , adding , disrupting or changing tions ofbase sequences. Each repetition is followed by short the sequence of specific genes ) and gene regulation in many segments of " spacer DNA ” from previous exposures to a species . By delivering the Cas9 protein and appropriate virus . CRISPRs are found in approximately 40 % of guide polynucleotides ( e . g . , guide RNAs) into a cell , the sequenced bacteria genomes and 90 % of sequenced archaea . organism ' s genome can be cut at a desired location . This CRISPRs are often associated with Cas or similar genes that system has recently been found to be adaptable to many code for endonucleases related to CRISPRs. The CRISPR / organisms including mammalian cells , fruit flies, and plants . Cas system is a prokaryotic immune system that confers The broad adaptability of this system has led to significant resistance to foreign genetic elements such as plasmids and strides in refining this system and the generation of many phages and provides a form of acquired immunity . CRISPR applications . The present invention may be applied to flies , spacers recognize and cut these exogenous genetic elements mosquitoes, human cells , and plants , for example . The in a manner analogous to RNAi in eukaryotic organisms. present invention provides methods and constructs for gen Improved methods and compositions for use in eukaryotic erating and neutralizing homozygous germline transmissible cells and organisms are needed for improved genomic mutations . engineering technologies . [0008 ] The invention provides in certain embodiments , a method for autocatalytic genome editing comprises genomi SUMMARY OF THE INVENTION cally integrating a construct comprising four elements : ( 1 ) a [0004 ] The present invention discloses methods and com gene encoding an endonuclease ( e . g . , Cas9 protein ) , ( 2 ) a positions for selectively introducing and / or neutralizing the sequence encoding one or more guide polynucleotides ( e . g . , spread of Mutagenic Chain Reaction (MCR ) elements from guide RNAs ) , ( 3 ) an effector cassette , and ( 4 ) homology organisms carrying them that do not affect organisms lack arms flanking the above three transgenes that target insertion ing such elements . of those elements ( 1 - 3 ) into the genome at the site deter [ 0005 ] MCR for autocatalytic genome editing is based on mined by the sequence flanking the guide polynucleotide ( s ) genomic integration of an MCR construct containing mul ( e . g . , guide RNA ( s )) ( element 2 ) . tiple elements . In some embodiments , the MCR invention [ 0009 ] In certain embodiments , the guide polynucleotide either : a ) injects the MCR construct as a DNA plasmid into ( e . g ., guide RNA ) once expressed binds to Cas9 protein and the germline of an organism and obtains transgenic organ directs sited directed cleavage of the genome at a specific isms carrying this insertion on one copy of a chromosome site . from which it can spread to the other chromosome ( creating [0010 ] In certain embodiments , the sequence encoding potential homozygous mutations ) as well as propagating the one or more guide polynucleotides is under a control of a same mutation via the germline to nearly all its offspring , or separate promoter. In certain embodiments , the separate b ) introduces the MCR construct into somatic cells in an promoter is an RNA -polymerase - I or III promoter . organism ( e . g . , using a plasmid or viral expression vector ) such that the construct spreads to other cells within that [ 0011 ] In certain embodiments , the construct is injected as organism . Therefore , the MCR provides an autocatalytic a DNA plasmid into a germline of an organism to obtain a method to generate homozygous mutations that propagate transgenic organism . with high fidelity via the germline to nearly